Protective role of vitamin B6 (PLP) against DNA damage in Drosophila models of type 2 diabetes

Growing evidence shows that improper intake of vitamin B6 increases cancer risk and several studies indicate that diabetic patients have a higher risk of developing tumors. We previously demonstrated that in Drosophila the deficiency of Pyridoxal 5′ phosphate (PLP), the active form of vitamin B6, causes chromosome aberrations (CABs), one of cancer prerequisites, and increases hemolymph glucose content. Starting from these data we asked if it was possible to provide a link between the aforementioned studies. Thus, we tested the effect of low PLP levels on DNA integrity in diabetic cells. To this aim we generated two Drosophila models of type 2 diabetes, the first by impairing insulin signaling and the second by rearing flies in high sugar diet. We showed that glucose treatment induced CABs in diabetic individuals but not in controls. More interestingly, PLP deficiency caused high frequencies of CABs in both diabetic models demonstrating that hyperglycemia, combined to reduced PLP level, impairs DNA integrity. PLP-depleted diabetic cells accumulated Advanced Glycation End products (AGEs) that largely contribute to CABs as α-lipoic acid, an AGE inhibitor, rescued not only AGEs but also CABs. These data, extrapolated to humans, indicate that low PLP levels, impacting on DNA integrity, may be considered one of the possible links between diabetes and cancer.

Growing evidence associates micronutrients as minerals and vitamins to genome integrity maintenance showing that their deficiency can damage DNA analogously to common carcinogens. Micronutrients act as co-factors or substrates for enzymes that counteract genotoxins as well as enzymes working in DNA metabolism (reviewed in 1 ). Vitamins belonging to B group as folates (vitamin B9 and its derivatives) and B12 vitamin act as cofactors of enzymes involved in the conversion of dUMP in dTMP by the transfer of a methyl group. It has been shown that in human cells and rodents 2 the impairment of this reaction due to folate and B12 deficiency causes Uracil misincorporation, a mutagenic lesion leading to point mutation, micronuclei formation and chromosome aberrations (CABs) that are known to contribute to the genesis and progression of human cancers [3][4][5] . Another consequence of impaired dUMP conversion is nucleotide pool imbalance that can compromise crucial cellular processes as DNA synthesis and repair. Impairment of DNA repair has been observed in cultured cells and animal models deprived of folates (reviewed in 6 ). Thus, it is not surprising that low folate levels have been correlated with some cancers as demonstrated by many epidemiological studies [7][8][9][10] .
Pyridoxal-5′-phosphate (PLP), the active form of vitamin B6, plays a role in protecting cells from DNA damage. Mammals produce PLP from vitamers (pyridoxine, pyridoxamine and pyridoxal), which are introduced by the diet and recycled in a "salvage pathway" through the action of two enzymes, Pyridoxal kinase and Pyrodoxine/pyridoxamine oxidase. PLP serves as a co-enzyme in approximately 160 enzyme reactions that regulate metabolism of glucose, lipids, amino acids, heme, DNA/RNA and many neurotransmitters [11][12][13] . In addition to work as cofactor, vitamin B6 plays antioxidant roles by counteracting the formation of genotoxic compound, the Advanced Glycation End-products (AGEs) 14,15 and by quenching oxygen reactive species 16,17 . Given its role in numerous metabolic reactions PLP has been associated to many human disorders including diabetes and cancer but underlying mechanisms are not fully elucidated (reviewed in 14,[18][19][20]. High expression level of Pyridoxal kinase (PDXK) has been positively correlated with survival of nonsmall cell lung cancer (NSCLC) patients 21 . In addition, Vitamin B6 intake and blood PLP levels were inversely correlated with the colorectal cancer risk 22 .
We recently demonstrated in Drosophila that PLP deficiency caused either by mutations in the pyridoxal kinase-coding gene (dPdxk) or by vitamin B6 antagonists results in chromosome aberrations (CABs) suggesting a role for Pyrodoxal kinase in chromosome integrity maintenance. This function is evolutionarily conserved in human and yeast as PDXK depletion by RNA interference in HeLa cells induces CAB formation 23 and mutations in BUD16 gene, encoding Saccharomyces cerevisiae Pyridoxal kinase, result in gross chromosome rearrangements 24 . More interestingly CAB frequency in Drosophila and human PLP-depleted cells was strongly enhanced by glucose treatment. In addition, PLP deficiency increases glucose content in dPdxk 1 mutant hemolymph and causes AGE accumulation in neuroblasts. Treatment of dPdxk 1 mutant cells with α-lipoic acid (ALA), a well-known AGE inhibitor, lowers both AGE formation and CAB frequency, suggesting a possible AGE-CAB cause-effect relationship. These findings indicate that a high intracellular glucose level has a dramatic clastogenic effect if combined with PLP deficiency and have prompted us to investigate if in diabetes low PLP levels can impair DNA integrity. Epidemiological studies clearly indicate that diabetes increases the risk of many types of cancer with mechanisms not yet fully understood. Possible links between these two diseases are hyperinsulinemia, hyperglycemia and fat-induced chronic inflammation. Several lines of evidence indicate that hyperglycemia may lead to cancer through DNA damage (reviewed in 25 ). It is well known that high glucose causes DNA damage by increasing the oxygen reactive species production via several mechanisms (namely the polyol pathway, hexosamine pathway, AGE pathway, and protein kinase C) 26,27 . Interestingly, oxidative damage and DNA strand breaks have been found in both type 1 and type 2 diabetic patients [28][29][30] .
To investigate if in diabetic cells PLP deficiency can impair genome integrity we used Drosophila as a model system. For the past decade Drosophila turned out to be a promising organism to model diabetes as flies and humans largely share mechanisms underlying the maintenance of balance between stored and circulating glucose (reviewed in 31 ). Insulin pathway signaling, a biochemical pathway well conserved in flies (reviewed in 32 ), plays a crucial role in glucose homeostasis maintenance as it triggers the uptake of glucose into liver, adipose tissue and muscle and promotes the storage of these nutrients in the form of glycogen. In humans and mice insulin pathway impairment, due to mutations, cause severe insulin resistance syndromes and type 2 diabetes (reviewed in 33 ). Also in Drosophila malfunctioning of insulin signaling, induced either by mutations or by feeding flies with a sugar rich diet, produce diabetic hallmarks that mimic type 2 diabetes (reviewed in 34 ).
In this work, we generated two different diabetic fly models, the first by depleting three well conserved proteins involved in insulin pathway as InR, Chico (IRS) and Akt1 and the second by rearing flies in sugar rich diet 35 . We showed that PLP deficiency in both models of diabetes caused severe chromosome and DNA damage, probably through AGE accumulation in turn caused by high glucose levels.
These results allowed to generalize the concept that PLP deficiency combined with high glucose strongly impairs genome integrity, suggesting that in diabetic patients low PLP levels may represent a cancer risk factor.

Results
Generation of diabetic fly models. We previously demonstrated that low PLP levels, due to mutations in the encoding Pyridoxal kinase gene (dPdxk), cause CABs and increase glucose content in larval hemolymph 23 . We provided evidence that these phenotypes are linked because high glucose causes AGE accumulation which in turn is responsible for CAB phenotype 23 . Starting from these results, we asked whether low vitamin B6 levels could be genotoxic in diabetes. To answer to this question, we generated two fly models of type 2 diabetes (reviewed in 31,34 ) using two strategies. In the first we silenced three evolutionarily conserved genes encoding proteins working in the insulin signaling pathway: Insulin receptor (InR), Insulin receptor substrate (Chico/IRS) and Akt1 (reviewed by 32 ). In the second we reared wild type larvae on a high sugar diet (HSD) according to Musselman et al. 35 . To deplete InR and Chico we performed in vivo RNA interference (RNAi) in fat body (using the ppl-Gal4 driver), an organ analogue to vertebrate adipose tissue and liver, crucial for nutrient storage and energy metabolism. Targeted gene knockdown was detected by reverse transcriptase-PCR (see Supplementary Fig. S1). The effects of Akt1 depletion were evaluated using the hypomorphic late lethal allele Akt1 04226 and all experiments reported in this paper were conducted on hemizygous individuals (Akt1 04226 /Df) to detect stronger phenotypes. Although in flies the most abundant circulating sugar is trehalose (a disaccharide formed by two glucose units) we focused on glucose as trehalose is a stable molecule and a non-reducing sugar 36 . In addition, evidence showed that glucose but not trehalose levels are regulated by mechanisms similar to those of higher organisms 37 . Glucose content in hemolymph from InR RNAi , chico RNAi Akt1 04226 /Df and HSD fed larvae was increased with respect to wild type and comparable to that exhibited by dPdxk 1 positive control 23 (Fig. 1A). In addition, InR and Chico RNAi-induced depletion, as well as HSD feeding, resulted in small size adults, a fly hallmark of insulin signaling impairment [38][39][40] ( Fig. 1B-D; Supplementary Fig. S2). It was not possible to analyze body size in Akt1 04226 /Df individuals as they die as third instar larvae.

Glucose treatment induces CABs in diabetic cells.
Evidence shows that glucose induces chromosome damage 41,42 . Thus, we first asked whether the high glucose content present in InR, Chico and Akt depleted individuals and also in HSD fed larvae affected DNA integrity. DNA damage was evaluated in two different ways. In the first we examined chromosomes from Akt1 04226 /Df, chico RNAi and HSD fed third instar larvae in colchicine treated brain preparations ( Fig. 2A), in the second, because of the low mitotic index induced by InR silencing, we examined γ-H2Av foci in InR RNAi brains fixed and immunostained with the pS137 anti-phospho-histone antibody that specifically recognizes γ-H2Av 43 (Fig. 2C). γ-H2AV is the homolog of the mammalian γ-H2AX and represents a suitable marker to study DNA damage since it marks the DSBs 44 , which are known to be at the basis of CABs 45 .
Neuroblasts from chico RNAi and HSD fed larvae exhibited frequencies of cells with broken chromosomes comparable to that of controls (0.5% and 0.3% respectively vs 0.44%) (Fig. 2B). Akt1 04226 /Df cells displayed a small but significant CAB frequency (1.86%) (Fig. 2B). In this case CABs are not due to the high endogenous glucose content since Akt1 04226 /Df mutants displayed hemolymph glucose levels comparable to those exhibited by chico RNAi and HSD fed larvae (Fig. 1A).
Since we previously showed that sugars such as glucose, fructose and sucrose strongly increased CAB frequency in dPdxk 1 neuroblasts 23 , we incubated brains from Akt1 04226 /Df, chico RNAi and HSD fed larvae in 1% glucose and we tested them for CABs. In all examined cases ( Fig. 2A,B) we found CABs ranging from 4.6% to 6.6%; in contrast glucose did not affect chromosome integrity in wild type neuroblasts, indicating that only diabetic cells are sensitive to extra sugar. Glucose treatment generated complex rearrangements; besides chromatid and isochromatid deletions ( Fig. 2A2-A4), we observed dicentric chromosomes accompanied by acentric fragments (formed when two broken non homologous chromosomes join together end to end) and also cells containing more than one rearrangement ( Fig. 2A5-A8). Glucose induced DNA damage also in InR depleted cells. While InR RNAi untreated larvae showed a percentage of γ-H2Av positive nuclei (4.5%) comparable to controls (4%), glucose treatment significantly increased this frequency (11.7% vs 4.3% in controls) (Fig. 2C,D).
Together these data indicate that the addition of extra glucose to brains from chico RNAi Akt1 04226 /Df, InR RNAi and HSD fed larvae causes chromosome damage differently than wild type, suggesting that diabetic cells are sensitive to glucose for CAB formation.

PLP deficiency induces high levels of DNA damage in diabetic cells. Our primary aim was to
verify whether it is possible to generalize the assumption that a simultaneous occurrence of high glucose and low PLP affects genome integrity. Thus, we examined chromosome and DNA damage in our diabetic models deprived of PLP. To deplete PLP we employed two strategies: in the first we fed Akt1 04226 /Df, InR RNAi , chico RNAi and HSD reared larvae with a strong PLP antagonist, the 4-Deoxypyridoxine (4-DP), and in the second we generated Akt1 04226 dPdxk 1 double mutant individuals performing genetic crosses. 4-DP caused a substantial increase in chromosome breakage in chico RNAi (~83-fold) Akt1 04226 /Df (~55-fold) and HSD larvae (~64-fold) (Fig. 3A,B) clearly indicating that PLP deficiency strongly impacted on chromosome integrity in both diabetes models. A significant, although lower, CAB increase (~24-fold) were also found in wild type 4-DP treated cells in agree with the PLP role in genome integrity maintenance, already demonstrated by our and other studies 23,24 . 4-DP enhanced CAB complexity more than glucose and produced cells with extensive chromosome fragmentation (Fig. 3A). As exactly quantifying the number of CABs in these cells was difficult, we decided to arbitrarily assign only five CABs to each cell with multifragmented chromosomes. As a consequence, CAB percentage reported in Fig. 3B may represent an underestimation. 4-DP also enhanced the frequency of γ-H2Av positive cells in InR RNAi larvae (24 vs 9.8% in control) (Fig. 3C,D). Interestingly most of 4-DP treated nuclei presented a foci pattern very similar to X rays-treated cells 46 with a mean of about 40 foci per cell that is consistent with the complexity of chromosome rearrangements observed in 4-DP treated brains from chico RNAi , Akt1 04226 /Df and HSD fed individuals.
The examination of Akt1 04226 dPdxk 1 brain cells revealed a CAB frequency (23%) higher than the sum of the frequencies displayed by each single mutant (Akt1 04226 1.1%; dPdxk 1 5%) (Fig. 3E). These data suggest a strong synergistic interaction between dPdxk 1 and Akt1 04226 mutations confirming the hypothesis that CABs originate by a combination of high glucose and low vitamin B6 levels accordingly with results obtained with 4-DP.
Based on the consideration that dPdxk 1 mutants contain more glucose than wild type strain we asked if PLP depletion could further increase glucose level in larval hemolymph from Akt1 04226 /Df, InR RNAi , chico RNAi and HSD fed individuals. As showed in Fig. 3F, 4-DP fed larvae exhibited a small but significant glucose content increase, with respect to untreated individuals.
Taken together these results indicate that in diabetic cells PLP deficiency strongly impairs DNA integrity.
AGEs contribute to DNA damage occurring in diabetic cells deprived of PLP. It is well known that PLP counteracts the accumulation of AGEs, genotoxic compounds abundant in most cells and fluid of diabetic patients 14,15,47 . AGEs originate from non-enzymatic amino group glycations of proteins, lipids and nucleic acids 48,49 , that accumulate during senescence and in high glucose conditions and their formation have been associated to reactive oxygen species production 26 . To investigate the mechanism through which PLP protects from damage in high glucose conditions we immunostained untreated, 1% glucose and 4-DP treated brains from InR RNAi , Chico RNAi and Akt1 04226 /Df larvae using a human antibody against AGEs. We did not test HSD larvae for AGEs since we expected that there is the same mechanism at the basis of CAB formation in both models of diabetes. InR, Chico or Akt1 depleted neuroblasts showed AGE accumulation (Fig. 4A,B) more pronounced after glucose treatment. 4-DP feeding further increased the frequency of cells positive to the anti-AGE staining in all examined cases (Fig. 4A,C). We next incubated Akt1 04226 /Df, InR RNAi and chico RNAi brains in 10 mM α-lipoic acid (ALA), an antioxidant compound that counteracts AGE formation and cooperates with PLP in ameliorating insulin resistance in prediabetic rats 50,51 . In all cases ALA treatment decreased AGE accumulation in both glucose and 4-DP treated cells (Fig. 4B,C). More interestingly ALA also decreased DNA damage. As showed in Fig. 5A, in Akt1 04226 /Df, and chico RNAi cells ALA treatment reduced CAB frequency to control values in both glucose treated and 4-DP fed larvae. Similarly, ALA also lowered the percentage of InR RNAi γ-H2Av positive cells in neuroblasts from glucose treated and 4-DP fed larvae (Fig. 5B). These results suggested a cause effect relationship between AGEs and CABs and indicated that AGEs are largely responsible for chromosome and DNA damage occurring in diabetic PLP depleted cells.

PLP rescues glucose-induced DNA damage in diabetic cells.
We previously demonstrated that PLP supplementation in both untreated and glucose treated dPdxk 1 mutants rescued AGE accumulation confirming the role of vitamin B6 in counteracting these compounds also in Drosophila 23 . Based on these findings, we asked if PLP supplementation could reduce chromosome and DNA damage in Akt1 04226 /Df, chico RNAi and InR RNAi glucose treated brains. Thus, we reared mutant/RNAi larvae in food supplemented with 10 −2 M PLP and examined them for CABs or γ-H2Av foci. To ascertain the PLP specificity we also tested brains from PLP fed individuals bearing twins (tws) mutation that causes a high frequency of CABs (about 40%) impairing a pathway uncorrelated with glucose metabolism 46 . PLP rescued glucose induced chromosome breakage in Akt1 04226 /Df and chico RNAi cells (Fig. 6A) but it did not influence CAB frequency in tws 430 mutant brains (Fig. 6B) confirming its specificity. PLP also rescued the percentage of InR RNAi glucose treated γ-H2Av positive cells (Fig. 6C). Taken together these data indicate that vitamin B6 improves glucose-induced DNA damage and strongly support the hypothesis that PLP is involved in chromosome integrity maintenance in high glucose conditions.
Remarkably PLP also rescued glucose content and body size in InR RNAi and Chico RNAi individuals suggesting that vitamin B6 plays a role in ameliorating the tissue sensitivity to insulin (Fig. 7A,B). These findings are consistent with our previous data 23 indicating that Pdxk 1 mutations cause a mild insulin resistance. Mechanisms linking PLP to insulin resistance will be the subject of future studies.

Discussion
For the past decade, Drosophila has been fast gaining popularity as a useful model system to study diabetes as it shares with humans mechanisms and molecules involved in hemolymph/blood glucose regulation. In Drosophila stable levels of circulating sugars are maintained by eight insulin-like proteins (Ilps) that are released in response to high glucose levels and by the adipokinetic hormone (AKH) that it is similar to glucagon and is released in response to low levels of circulating sugar [52][53][54][55] . Type 1 and 2 diabetes have been modeled in Drosophila reproducing hallmarks of both diseases 35,[56][57][58] giving the chance to isolate new genes and identify functional interactions that can be exploited for therapeutic interventions on human patients.
We demonstrated that PLP deficiency due to mutations in dPdxk Drosophila gene causes DNA damage and increases glucose content in larval hemolymph. We showed that these phenotypes are correlated and that high glucose is largely responsible for DNA damage. In addition we showed that also in human HeLa cells the relationship between PDXK, DNA integrity and glucose content has been maintained in the course of evolution 23 .
In the present work we have provided evidences that PLP deficiency in fly diabetic cells strongly impair DNA integrity. We demonstrated the combined genotoxic effect of low PLP and high glucose levels using two different diabetes models, the first obtained by downregulating the expression of three well conserved insulin pathway genes: InR, chico and Akt1, and the second obtained by feeding flies with a high glucose diet (HSD). Both insulin signal impairment and HSD feeding are considered promising strategies to model type 2 diabetes in flies 34 . Glucose levels obtained by depleting InR, Chico or Akt1 were similar to those obtained by Ugrankar et al. 37 , although we used a different fat body driver to silence InR and chico and also a different Akt1 allele.
We showed that high level of endogenous glucose caused by either insulin signaling impairment or HSD feeding is not sufficient to compromise DNA integrity. A possible explanation of this can be that fly metabolism relies mainly on treahalose that represents the more abundant circulating not-reducing sugar with ratio 100:1 with respect to glucose. Thus, to observe glycation effects the system need to be forced by adding more glucose. We showed in fact that glucose treatment in Inr, Chico or Akt1 depleted brains as well as in neuroblast from HSD fed larvae caused CABs or γ-H2Av foci. In contrast glucose did not influence chromosome integrity in wild type cells suggesting that diabetic cells are more sensitive to glucose respect to wild type cells. We found that Akt1 04226 / Df mutants displayed a low frequency of CABs also in untreated cells. However, based on evidence indicating that the human AKT counterpart is involved in DNA integrity maintenance 59 we think that CABs in these cells are not related to glucose metabolism, but are due to an improper DNA repair.
More importantly, we demonstrated that in both our hyperglycemic models, DNA damage was exacerbated when PLP levels were decreased by 4-DP, a strong PLP antagonist. 4-DP caused a dramatic CAB increase in chico RNAi (83.6%), Akt1 04226 /Df (57.4%) and HSD larval brains (64%) and also enhanced about 5 times the percentage of γ-H2AV positive cells in InR RNAi individuals with respect to untreated counterparts. 4-DP further increased glucose content that, combined with an impaired antioxidant defense system, caused by PLP deficiency, may contribute to amplify chromosome and DNA damage. In addition we provided genetic evidence of a strong synergistic interaction between Akt1 and dPdxk 1 mutations in CAB formation in double mutant individuals, accordingly with results obtained using 4-DP.
These results demonstrate that it is possible to extend the concept that low PLP levels cause DNA damage in high glucose conditions also to other diabetic contexts different from those caused by Pdxk 1 mutation 23 .
Regarding the mechanism leading to CABs in diabetic PLP deprived cells we identified the pathway that leads to AGE formation as the best candidate that allow to correlate PLP, glucose and DNA damage. We showed that InR RNAi , chico RNAi or Akt1 04226 /Df diabetic cells accumulate AGEs which in turn are reduced by α-lipoic acid (ALA). More interestingly ALA also rescued both CABs and γ-H2Av foci suggesting a cause effect relationship between AGEs and CABs. As AGEs are known to be genotoxic through oxygen reactive species formation, these findings indicate that in diabetic cells PLP may safeguard genome integrity preventing AGEs from attack DNA. Our hypothesis is based on in vitro 15 and in vivo studies showing that PLP is able to counteract AGE formation. It has been reported that PLP inhibits AGE accumulation in streptozotocin-induced diabetic rats, preventing the progression of nephropathy 60 . In addition, in glomerular cell nuclei of these rats PLP reduced the levels of carboxyethyl-2′deoxyguanosine (CEdG), a DNA glycation product that can cause strand breaks 61 . The exact mechanism through which PLP counteracts AGEs is not to date fully elucidated. A hypothesis is that PLP may block AGE formation by trapping the 3 deoxyglucosone (3-DG), an intermediate product of AGE metabolism. It has been in fact indicated, by in vitro experiments, that PLP is able to sequestrate 3-DG in a dose dependent manner 62 . However also other mechanisms are possible. PLP also works as a cofactor of enzymes that convert dUMP in dTMP. dPdxk 1 mutants have an unbalanced amount of dUMP respect to dTMP 23 but as are not sensitive to replication block induced by HU, this excess of dUMP does not seem to be the principal mechanism that leads to CABs. Anyway, we cannot exclude that nucleotide unbalance caused by PLP depletion can slightly affect chromosome stability in our diabetes models by influencing the rate of DNA repair. Furthermore, to the hypersensitivity of diabetic cells to 4-DP toxicity can contribute an impairment of DNA repair associated to diabetes. It has been demonstrated that diabetic patients showed, besides high levels of basal endogenous and oxidative damage, an increased sensitivity to DNA damaging agents combined to a decreased capability to repair damage induced by these drugs 63 .
Altogether our data, extrapolated to human, strongly support the hypothesis that DNA damage represents one of the causative factors linking diabetes to cancer. In addition, they highlight the crucial role of PLP in chromosome stability maintenance in diabetic cells suggesting that low PLP levels can represent a critical cancer risk factor for diabetic patients. Low PLP levels in normal physiology represent a rare condition due to excessive consumption of alcohol, drugs commonly used to treat pathologies as tuberculosis or arthritis (e.i. isonyazide, cycloserine, penicillamine) or celiac disease and renal dialysis 64 . However, interestingly, low PLP levels have been associated to diabetes in murine models and epidemiological studies [65][66][67] . In particular, a recent study 68 based on the examination of three groups of individuals, diabetic, diabetic with nephropathy and healthy subjects, showed that respectively 63% and 58% of diabetic patients with and without nephropathy presented low PLP plasma levels (<30 nM/L). Moreover, altered levels of PLP precursors pyridoxine and pyridoxamine have been found in diabetic patients suggesting an impaired PLP metabolism associated to diabetes. At the light of these considerations our findings suggest that in diabetic individuals PLP levels should be kept under control in order to limit the risk of DNA damage.

Methods
Drosophila strains and crosses. InR v992 and chico v7776 lines were obtained from the Vienna Drosophila Resource Center (VDRC) stock center. dPdxk 1 mutation has been described previously 23 . ppl-Gal-4 driver 52 , Akt1 04226 and Df Exel 7328 (89A12-89B6) lines were obtained from the Bloominghton Stock Center. InR RNAi and chico RNAi flies were generated by crossing females bearing the RNAi construct to males carrying the fat body specific ppl-Gal4 driver. Mutations and deficiencies were kept in stock over the TM6C Sb, Tb balancer; homozygous and hemizygous mutant larvae were recognized for their non-Tubby phenotype. The Oregon-R strain was used as wild-type control.
Double mutants dPdxk 1 Akt1 04226 were generated by recombination by crossing Akt1 04226 /TM6C females to dPdxk 1 /TM6C males. Akt1 04226 /dPdxk 1 females resulting from this cross were mated to Ap Xa /TM6C males. Single males resulting from this cross were mated to Ap Xa /TM6C females. From these crosses were established about 60 stocks crossing inter se males and females carrying recombinant chromosomes balanced over TM6 C. The presence of both mutations on the recombinant chromosomes was verified by complementation analysis. The balancers and the genetic markers used in these crosses are described in detail in FlyBase (http://flybase.bio. indiana.edu/). Fly food recipes. All flies, from embryo stage, were raised on a standard food. To induce insulin resistance wild type larvae were reared on a sugar rich medium which is a standard food with an increased sucrose concentration. 100 ml food contains: agar (0.68 g), yeast (6.52 g), flour (3 g) propionic acid (600 µl) and sucrose (5.13 g = 0.15 M for standard food; 34.2 g = 1.0 M for high sugar diet, HSD). Chromosome cytology. Colchicine-treated larval brain metaphases for CAB scoring were obtained as previously described 69 . For immunostaining, brains from third instar larvae were dissected and fixed as described in Bonaccorsi et al. 70 . Brain preparations were rinsed in PBS 0.1% Triton (PBST), incubated overnight at 4 °C with primary antibodies diluted in PBST, rinsed in PBST, and then incubated for 1 hr at room temperature with the secondary antibody. The primary antibodies used were: rabbit anti-Histone H2AvD pS137 (1:100 in PBST; Rockland code #600-401-914) and rabbit anti-human AGE antibody (1:200 in PBST; ab23722, Abcam, UK). Both antibodies were detected with Alexa-Fluor-555-conjugated anti-rabbit antibody (1:300 in PBST; Molecular Probes, Eugene, OR). All fixed preparations were mounted in Vectashield H-1200 with DAPI (Vector Laboratories, Burlingame, CA) to stain the DNA. Observations were carried out using a Zeiss Axioplan fluorescence microscope equipped with CCD camera (Photometrics CoolSnap HQ).

Drug treatments.
To evaluate the effects of PLP inhibitor 4-deoxypyridoxine (4-DP) on CABs and γ-H2Av foci, flies were allowed laying eggs on a fly standard growth medium for 5 days. Then, adults were removed and 4-DP 2 mM was added to medium (containing first and second instar larvae). 5 days later third instar larvae were dissected and brains fixed. For chromosome preparations brains were incubated 90 min in colchicine before fixing. To detect γ-H2Av foci brains were treated according to immunostaining procedure.
To test the effects of glucose, PLP, and α-lipoic acid (ALA) on CABs and γ-H2Av foci, brains were dissected from third instar larvae and incubated in 2 ml of saline supplemented with 10% fetal bovine serum (FBS, Corning) for 4 hours with or without addition of 1% glucose, 1 mM PLP or 10 mM ALA. For chromosome preparations 1 h before fixation brains were treated with colchicine 10 −2 M (final concentration). For γ-H2Av foci detection brains were treated according to immunostaining procedure.
Picture acquisition. Pictures of adult flies were taken using a Nikon D5200 digital camera mounted on a stereomicroscope (Nikon SMZ-1). Pictures were taken using a 1/6 second exposure, and 800 iso.
Glucose and wing measurement. Glucose concentration in Drosophila hemolymph was measured using the Infinity Glucose Hexokinase reagent (Thermo scientific). Hemolymph collection and glucose measurement were done as described in Marzio et al. 23 .
For wing analysis, flies were anesthetized with CO 2 . Dissected wings were placed in absolute ethanol, and mounted in a 6:5 mixture of lactic acid/ethanol 71 . Measurements were made directly on digitized images of mounted wings using ImageJ software.  InR F GTGCTCCTCCGGTCTTATCGA InR R GTGACGTTCAGCATAGCGGAG rp49 F TACAGGCCCAAGATCGTGAA rp49 R ACGTTGTGCACCAGGAACTT Statistical analysis. Results are expressed as means ± SEM; probability values < 0.05 were considered statistically significant. Statistical analysis of the data was done with the two-tailed Student's t-test. Data availability. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


s2fieldsofstudy: Biology Medicine

text:
Cardiac ischemia/reperfusion injury is inversely affected by thyroid hormones excess or deficiency in male Wistar rats

Aim Thyroid dysfunctions can increase the risk of myocardial ischemia and infarction. However, the repercussions on cardiac ischemia/reperfusion (IR) injury remain unclear so far. We report here the effects of hypothyroidism and thyrotoxicosis in the susceptibility to IR injury in isolated rat hearts compared to euthyroid condition and the potential role of antioxidant enzymes. Methods Hypothyroidism and thyrotoxicosis were induced by administration of methimazole (MMZ, 300 mg/L) and thyroxine (T4, 12 mg/L), respectively in drinking water for 35 days. Isolated hearts were submitted to IR and evaluated for mechanical dysfunctions and infarct size. Superoxide dismutase types 1 and 2 (SOD1 and SOD2), glutathione peroxidase types 1 and 3 (GPX 1 and GPX3) and catalase mRNA levels were assessed by quantitative RT-PCR to investigate the potential role of antioxidant enzymes. Results Thyrotoxicosis elicited cardiac hypertrophy and increased baseline mechanical performance, including increased left ventricle (LV) systolic pressure, LV developed pressure and derivatives of pressure (dP/dt), whereas in hypothyroid hearts exhibited decreased dP/dt. Post-ischemic recovery of LV end-diastolic pressure (LVEDP), LVDP and dP/dt was impaired in thyrotoxic rat hearts, whereas hypothyroid hearts exhibited improved LVEDP and decreased infarct size. Catalase expression was decreased by thyrotoxicosis. Conclusion Thyrotoxicosis was correlated, at least in part, to cardiac remodeling and increased susceptibility to IR injury possibly due to down-regulation of antioxidant enzymes, whereas hypothyroid hearts were less vulnerable to IR injury.

Introduction Thyroid hormones (TH) are known to play crucial roles in the regulation of cardiovascular system homeostasis. By binding to nuclear receptors, TH regulates the expression of several contractile and calcium-handling proteins, ion channels and sympathetic tonus [1]. As a result, cardiac inotropic, chronotropic, lusitropic and dromotropic properties, as well as vascular resistance, are directly affected by TH levels.
It has been widely recognized from clinical data that thyroid dysfunction is correlated to increased cardiovascular morbidity and mortality [2]. Overall, tachycardia and tachyarrhythmic events, cardiac hypertrophy and heart failure have been frequently reported in conditions of TH excess, such as Graves' disease, thyroid or pituitary adenoma, and toxic multinodular goitre [3,4]. On the other hand, bradyarrhythmias, mild hypertension, impaired systolic and diastolic functions have been associated to TH deficiency [4,5].
Importantly, clinical reports have demonstrated that hyperthyroid and hypothyroid patients are more predisposed to myocardial ischemia and acute myocardial infarction (AMI), the leading cause of death among cardiovascular diseases, when compared to euthyroid patients [6,7]. In case of TH excess, coronary artery spasm and increased prothrombotic state can elicit coronary artery occlusion and myocardial ischemia [8][9][10]. Myocardial ischemia can be further worsened by increased cardiac workload and oxygen demand elicited by TH excess [11]. In hypothyroid patients, dyslipidaemia and increased circulating cholesterol levels are the main contributors to the formation of atherosclerotic plaque and development of coronary artery disease [3,10]. Nonetheless, decreased blood vessel density and increased arterial wall stiffness account for the declined coronary blood flow and the higher risk of myocardial ischemia, despite the decreased cardiac workload induced by TH deficiency [11][12][13].
Reperfusion remains the most effective therapeutic maneuver to rescue ischemic myocardium and has been widely recommended by both American Heart Association and European Society of Cardiology [14,15]. Even so, reperfusion per se recruits pro-apoptotic pathways that culminate in cardiomyocyte death and further infarct expansion, a condition referred to as reperfusion injury [16]. Among several pathophysiological mechanisms, Redox imbalance has been demonstrated to play a pivotal role in the progression of ischemia/reperfusion (IR) injury by promoting oxidative damage and cell apoptosis [17].
The clinical relevance of changes on TH level in the progression of IR injury and the repercussion on AMI outcomes are unclear, whereas experimental data remain conflicting so far. Administration of 3,5,3'-triiodothyronine (T3), the main cellular active thyroid hormone, has been reported to potentiate post-ischemic recovery of myocardial mechanical properties in experimental models of IR injury [18,19]. Conversely, IR-induced myocardial damage and ventricular arrhythmias have been shown to be attenuated in hypothyroid rat hearts in comparison to euthyroid and thyrotoxic rats [20,21]. Therefore, this study aimed to assess both cardiac mechanical properties, as well as the extent of myocardial damage induced by IR in rats exposed to long-term TH deficiency and excess in comparison to euthyroid rats. The potential role of antioxidant enzyme imbalance was also investigated.

Materials and methods Animals
This study followed the standards and ethical guidelines of the Ethics Committee for Research of the Federal Rural University of Rio de Janeiro and of the Federal University of Rio de Janeiro. It was approved by the Ethics Committee for Research of the Federal Rural University of Rio de Janeiro and of the Federal University of Rio de Janeiro under the number IBCCF194-07/16. Furthermore, all the standards proposed by the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health (NIH) Publication No. 85-23, revised 1996) were observed.

Experimental protocol
Male Wistar rats (8 weeks-old, 170-200 g) obtained at the Central Vivarium (Carlos Chagas Filho Institute of Biophysics, UFRJ, Brazil) were housed in cages under controlled temperature (21 ± 2˚C), daily exposed to 12-hour light-dark cycle (lights off at 7:00 pm) and water and standard chow ad libitum. The animals were divided into three groups: euthyroid (CTL, N = 18), hypothyroid (MMZ, N = 14) and thyrotoxic (T4, thyroxine group, N = 17). Hypothyroidism and thyrotoxicosis were induced by methimazole (300 mg/L) and L-thyroxine (12 mg/L), respectively, administered in drinking water for 35 days [22]. No animal exhibited adverse symptoms during all period of vehicle, T4 and MMZ exposure. After treatments, all animals were euthanized under anesthesia with isoflurane by exsanguination, and blood samples were collected for serum T4 and T3 measurements by radioimmunoassay. Hearts were excised and weighed, and the tibia length was measured for pathological analysis. The susceptibility to IR injury was evaluated in isolated hearts using a Langedorff apparatus. Cardiac samples were collected and stored at -80˚C for posterior molecular analyses.

Ex vivo IR experiments
Methods for isolated rat heart experiments were similar to those previously described [23]. Excised hearts were weighted and placed in modified Krebs-Henseleit solution (KHS) (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 10 mM glucose, 1.8 mM CaCl 2 , saturated with 95% O 2 and 5% CO 2 ). Aortas were hung on the cannula of a modified Langendorff apparatus and hearts were artificially perfused with modified KHS adjusted for pH 7.4 and 37˚C at a constant flow (10 mL/min). A latex balloon was inserted into the left ventricular (LV) chamber through an atrial incision to assess mechanical performance. Hearts were kept immersed in perfusion solution and baseline LV end-diastolic pressure (LVEDP) was set at 10 mmHg. After 20 min of baseline period, wherein heart rate, diastolic and systolic pressures were stable, the peristaltic pump was stopped and hearts were submitted to 30 min of global ischemia and subsequent 60 min of reperfusion. LV developed pressure (LVDP), LV systolic pressure (LVSP), LV end-diastolic pressure, LV maximal derivative of pressure (max. dP/dt) and LV minimal derivative of pressure (min. dP/dt) waveforms were recorded with a pressure transducer (PT 300, Grass Technologies). The transducer was connected to an amplifier (ML 110 ADInstruments), which was connected to an analogical/ digital converter (PowerLab 400, ADInstruments). All recordings were digitized and stored on a computer for later analysis using the program LabChart 5.0 (ADInstruments 1 ).

Measurement of infarct size
Ventricular sections were sliced into approximately 1.5 mm from apex to base and incubated in 1% (w/v) TTC in phosphate buffer (pH 7.4) for 5 min at 37˚C. All slices were placed in a 10% (v/v) formaldehyde solution for 24 h to improve contrast between stained (viable) and unstained (necrotic) tissues. Ventricle slices were placed between two glass slides and their images were digitally acquired in a scanner. Infarct size was determined using ImageJ software (NIH ImageJ: National Institute of Health, USA, version 1.22). Values were expressed as % of total ventricular area [24].

Radioimmunoassay for total serum T4 and T3
Blood samples were collected at the end of treatment after euthanasia (35 th day). Samples were centrifuged at 1931.9 xg for 20 min, and sera were separated and stored at -20˚C. Serum T3 and T4 were determined by specific Coated-Tube Radioimmunoassay kits (MP Biomedicals, LLC, USA). All the procedures were carried out following the kit recommendation.

Quantitative polymerase-chain reaction (qPCR)
To estimate mRNA expression levels of superoxide dismutase type 1 (SOD1), SOD2, catalase, glutathione peroxidase type 1 (GPX1) and GPX3 (Table 1), total RNA was extracted from LV tissue samples using RNeasy1 Fibrous Tissue Mini Kit (QIAGEN) and cDNA was prepared from 1 μg of total RNA using High-Capacity Reverse Transcription kit (Thermo Fisher Scientific) according to the manufacturer's instructions. mRNA levels of target genes (Table 1) were evaluated by qRT-PCR. Amplification reactions containing 1ng of cDNA were performed at 60˚C during the annealing and extension cycles. The expression of chosen genes was normalized to GAPDH as an internal control. The quantification of selected mRNA was determined by 2 -(ΔΔCT) method in a Viia7 Software v1.2.4 and expressed as fold change of MMZ and T4 group compared to the control group.

Statistical analysis
Data are presented as mean ± standard error of mean (S.E.M.). Normal statistical distribution of all data was determined by Shapiro-Wilks test (Statext v2.7, http://www.statext.com/index. php). One-way ANOVA followed by Bonferroni post-hoc test were used to compare MMZ and T4 groups to CTL group (Prism1, GraphPad). Statistical differences were considered significant when P < 0.05.

Results and discussion
The present study provides evidence that the pathophysiological progression of myocardial IR injury can be distinctly affected by TH excess or deficiency. While thyrotoxic rat-hearts were more vulnerable to post-ischemic myocardial stunning than euthyroid rat hearts, post-ischemic recovery of mechanical properties was improved and infarct size was decreased in hypothyroid rat hearts. Increased susceptibility to redox imbalance and oxidative damage might contribute to these deleterious effects, given that thyrotoxic rat-hearts exhibited decreased mRNA expression level of antioxidant enzymes SOD2 and catalase.
TH deficiency is the most commonly diagnosed thyroid dysfunction and can result from several different environmental and physiological disturbances, such as iodine deficiency, primary atrophic hypothyroidism and Hashimoto's thyroiditis [25]. The most frequent causes of TH excess are Graves' disease, toxic multinodular goitre and thyroid adenoma [25]. Despite the limitations to mimic all features of human thyroid dysfunctions, delivery of MMZ and T4 in drinking water have been frequently used as non-invasive experimental models of hypothyroidism and thyrotoxicosis, respectively [22]. MMZ inhibits thyroidal enzyme thyroperoxidase and incorporation of iodine into thyroglobulin, resulting in drop of T3 and T4 production, as evidenced in the MMZ group ( Table 2, P < 0.05 vs. CTL group). Unsurprisingly, long-term T4 exposure increased serum level of T4 ( Table 2, P < 0.01 vs. CTL group). In addition, serum T3 levels were also increased in the T4 group (Table 2, P < 0.01 vs. CTL group), given that T4 can be converted into T3 by deiodinases (DIO) type 1 and 2 in target tissues. Taken together, these findings confirm that MMZ and T4 groups developed hypothyroidism and thyrotoxicosis, respectively as expected. TH-deficient rats exhibited decreased mean body weight ( Table 2, P < 0.001 vs. CTL group), in agreement with the essential role of TH in the process of musculoskeletal growth by regulating pituitary and hypothalamic growth hormone synthesis [26]. Furthermore, TH deficiency has been correlated to decreased food intake and decline on body weight gain [26]. Conversely, body weight gain was not significantly affected by long-term T4 exposure ( Table 2, P > 0.05 vs. CTL group). Although previous data have demonstrated that food intake can be increased by TH excess, it has been widely recognized that metabolic rate can also be increased in such a condition, which might elicit balanced body weight gain [27].
Clinical and pre-clinical data have demonstrated that TH excess can induce substantial cardiac morphological changes [28,29]. Indeed, thyrotoxic rats exhibited increased HW ( Table 2, P < 0.01 vs. CTL group), HW/BW ( Table 2, P < 0.001 vs. CTL group) and HW/tibia length ( Table 2, P < 0.01 vs. CTL group) in comparison to euthyroid rats, suggesting development of cardiac hypertrophy. At initial phases, cardiac growth in response to TH involves proportional rates of cardiomyocyte enlargement and proliferation of other cell types, such as fibroblasts and vascular cells [30][31][32]. At the transcriptional level, contractile and calcium-handling proteins are up-regulated, resulting in a condition frequently referred as physiological cardiac hypertrophy [33,34]. As demonstrated elsewhere, TH can potentiate calcium-induced calcium release from sarcoplasmic reticulum, as well as cytosolic calcium concentration during systolic period by up-regulation of ryanodine receptor expression level [33][34]. In addition, up-regulation of sarcoplasmic Ca 2+ ATPase, as well as down-regulation of phospholambam expression levels can potentiate calcium uptake by sarcoplasmic reticulum, which has been correlated to increased relaxation rate during diastole, and further calcium release in the following excitation-contraction cycle [33][34]. Consequently, hyperthyroid patients can present increased fractional shortening and ejection fraction compared to euthyroid patients [35]. In keeping with these evidences, baseline contractility and relaxation properties were potentiated by TH excess, as evidenced by increased LVSP (  [36][37][38]. Even so, cardiac atrophy has been reported in few experimental clinical studies, whereas cardiac hypertrophy and dilation can be observed during the progression towards heart failure [39]. Corroborating these evidences, TH-deficient rats did not show significant changes in relative HW/BW and HW/tibia length (Table 3, P > 0.05 vs. CTL group) in comparison to euthyroid rats, although absolute HW was decreased (Table 2, P < 0.01 vs. CTL group). Furthermore, contractility (Table 3, P < 0.001 vs. CTL group) and relaxation (Table 3, P < 0.05 vs. CTL group) rates were significantly decreased by hypothyroidism, in agreement with the neuralgic role played by TH in the regulation of contractile and calcium-related proteins. Indeed, decreased fractional shortening, ejection fraction, stroke volume and cardiac output have been strictly correlated to TH-deficiency-related diseases [4,5].
After the onset of ischemia, LVEDP progressively rose in all experimental groups (Fig 1). Ischemic contracture, also known as stone heart, has been attributed to reduced ATP bioavailability. The shift towards glycolysis and the unbalanced metabolic demand result in increased lactate production and cytosolic acidification, an effect counterbalanced by the extrusion of H + by the Na + /H + exchanger (NHX) [40]. As a result of increased NHX-induced sodium influx, the activity of Na + /Ca 2+ exchanger (NCX) also increases, as does intracellular calcium concentration and LVEDP [41][42][43]. Interestingly, ischemic contracture was delayed (Fig 1F), whereas post-ischemic LVEDP reached the lowest levels among hypothyroid rat hearts (Table 4, P < 0.05 vs. CTL group). In agreement with our findings, Mourouzis et al. observed decreased post-ischemic LVEDP in hypothyroid rat hearts, which resulted in increased LVDP   relaxation velocities reached the lowest levels among thyrotoxic rat hearts (Table 4, P < 0.05 vs. CTL group). These findings suggest that TH excess not only worsened IR-induced myocardial contracture, but also impaired the recovery of contractility properties, a condition known as myocardial stunning. In contrast, acute and short-term exposure to T3 have been reported to improve IRinduced myocardial stunning in isolated rat hearts [18,19]. The increased glucose uptake induced by TH has been shown to elicit cardioprotection against doxorubicin toxicity, and this change might also elicit cardioprotection against IR injury, as evidenced by previous data [44]. At low doses or short-term exposure, TH might induce compensated cardiac hypertrophy and moderate increases on myocardial mechanical properties, consistent with TH positive inotropic and lusitropic effects [18]. However, long-term exposure to supraphysiological concentration of TH can elicit remarkable increases in ATP and oxygen consumption, which might turn thyrotoxic hearts more vulnerable to IR damage [45][46][47]. Conversely, hypothyroid hearts exhibited decreased metabolic rate which might be convenient in a condition marked by low bioavailability of oxygen and metabolic fuels such as ischemia [11,48]. Indeed, it has been postulated that the decreased myocardial conversion of T4 into T3 after AMI might be an adaptation to the new metabolic demand [49]. In keeping with this hypothesis, infarct size was decreased among hypothyroid rat hearts at approximately 45% compared to euthyroid rat hearts (Fig 2,   Repercussion of thyroid status on cardiac ischemia/reperfusion injury The pathophysiological mechanisms involved in the opposite effects induced by TH excess and deficiency in the progression of IR injury remain unclear. However, pre-clinical studies have correlated the regulation of cellular metabolism and mitochondrial respiratory chain activity to reactive oxygen species (ROS) production and oxidative damage elicited by TH excess [45,[50][51][52][53][54]. In a condition of redox imbalance, antioxidant enzyme provide defense against oxidative damage by promoting ROS clearance. Interestingly, catalase, SOD2 and GPX1 mRNA expression level (Fig 3) was down-regulated in thyrotoxic rat-hearts at 46.0% (P < 0.05 vs. CTL group), 37.6% (P > 0.05 vs. CTL group), and 51% (P > 0.05 vs. CTL group), respectively. On the other hand, SOD1 and GPX1 mRNA expression levels were up-regulated at 93.0% and 52.9% (Fig 3, P > 0.05 vs. CTL group), respectively, in hypothyroid rat-hearts. As previously demonstrated, SOD1, SOD2 and GPX knockout mice are more vulnerable to IRinduced damage and cell death [55][56][57]. On the other hand, overexpression of SOD and catalase have been correlated to post-ischemic mechanical improvements and decreased organ damage [58,59]. Previous data have also correlated decreased levels of GPX, coenzyme Q9 and Q10 to increased levels of lipid and proteins oxidative damage in thyrotoxic hearts [51][52][53][54]. Although myocardial oxygen level drops significantly after the onset of ischemia, residual oxygen level remains at approximately 3-5 Torr, which enables the production of ROS at sublethal concentration [60][61][62]. Nevertheless, abrupt reestablishment of myocardial oxygen level at the onset of reperfusion further increases ROS production [60]. At high concentrations, ROS can induce oxidative damage and contribute to the opening of mitochondrial permeability transition pore and release of pro-apoptotic molecules, contributing to infarct expansion [17,63]. Conversely, experimental studies have demonstrated that mitochondrial ROS production and leak at complexes I and III are reduced in hypothyroid hearts, resulting in protection against oxidative damage [45,64,65]. Therefore, redox status might play a pivotal role in the switch between increased and decreased susceptibility to myocardial IR injury elicited by TH excess and deficiency, respectively.
It remains unclear, though, how cardiac antioxidant defense was down-regulated by thyrotoxicosis. Clinical studies have reported decreased antioxidant enzymes expression levels in hyperthyroid patients, which can be restored by antithyroid drug therapy [66,67]. However, several data have demonstrated that hyperthyroidism can be followed by increased expression levels of antioxidant enzymes, as a compensatory mechanism to increased ROS production [51][52][53][54]. Noteworthy, thyrotoxic rat-hearts were also exposed to IR in the present study, suggesting that thyrotoxicosis might be followed by decreased expression levels of antioxidant enzymes particularly in conditions of physiological challenge. In addition, these findings also suggest that secondary effects, instead direct transcriptional effect of TH receptors, might play important roles in thyrotoxicosis-induced redox imbalance. Indeed, it has been demonstrated that hyperthyroidism can elicit autonomic imbalance, severe insulin resistance and unbalanced adipokine bioavailability, including decreased circulating levels of vaspin, while visfatin and resistin levels can be increased, irrespective of changes on body weight [68][69][70]. Together, these metabolic abnormalities can elicit cardiomyocyte pro-inflammatory changes and mitochondrial dysfunctions, which can ultimately aggravate redox imbalance and IR damage [71,72].
Taken together, the present findings demonstrated that long-term exposure to TH excess or deficiency distinctly affected the progression of myocardial IR injury. Whereas post-ischemic recovery of mechanical properties was slightly improved and infarct size decreased by TH deficiency, myocardial stunning was worsened was increased in thyrotoxic rat-hearts. These findings were correlated to decreased expression of catalase in the condition of TH excess. However, additional studies will be necessary to investigate the role of redox imbalance and cardiac remodeling in the opposite effects induced by TH deficiency or excess in the susceptibility to IR injury.


extfieldsofstudy: Chemistry Medicine

text:
Unique Responses of Hepatocellular Carcinoma and Cholangiocarcinoma Cell Lines toward Cantharidin and Norcantharidin

The present study aimed to investigate whether cell lines from human gastric and liver cancers respond differently toward cantharidin (CTD) and norcantharidin (NCTD) than other types of cancer cells. We first established the half maximal inhibitory concentrations (IC50s) of CTD for a large panel of cancer cell lines representing the 12 major types of human cancers and the mode of cell death induced by the two compounds. We next compared the growth inhibitory effects as well as the corresponding modes of action of CTD and NCTD. The IncuCyte ZOOM system was used as a semi-high throughput means to define IC50s and 90% inhibitory doses (IC90s) as a reference for the maximal tolerable doses (MTDs) for the two compounds in 72 cancer cell lines. Classical clonogenic survival assay was used to assess the anti-proliferative effect of CTD on selected cell lines of interest. In addition, DNA content-based flow was used to interrogate the modes of cell death following CTD or NCTD exposure. The results of these experiments led to several findings. 1). Cell lines representing hepatocellular carcinomas (HCCs) and cholangiocarcinomas (CCs) were among the most sensitive toward CTD, consistent with the previous clinical study of this compound and its source of origin, Mylabris. 2). Among the individual cell lines of a given cancer types, the sensitivity trends for CTD and NCTD did not exhibit a good correlation. 3) CTD and NCTD caused distinctive cytotoxic effects on HepG2 cells. Specifically, while a cytostatic effect is the primary cause of growth inhibition of CTD, cytotoxic effect is the main contributing factor for the growth inhibition of NTCD. These results indicate that liver cancer cell lines are among the most sensitive to CTD and that CTD and NCTD exhibit their effects through distinct mechanisms.

Introduction
Liver cancers represent one of the major types of cancers worldwide. In particular, hepatocellular carcinomas (HCCs) and Cholangiocarcinomas (CCs) together account for the majority of the cancer incidents in the liver. Sorafenib and Regorafenib are currently the only drugs approved by the US FDA to treat HCCs [1,2] , though the effectiveness of these drug is limited [3] . No drugs have been approved to treat CCs. Thus, effective drugs for HCC and CC are still in urgent need.
Historically, Mylabris has been reported to be effective for treating liver cancers and gastro-Ivyspring International Publisher esophageal cancers in China [3] . Moreover, modern clinical studies have showed that cantharidin (CTD), the presumed active antitumor compound of Mylabris, was effective in treating HCCs and gastro-esophageal cancer [4] , despite the fact that both Mylabris and CTD are potent poison [4] . Remarkably, it is generally accepted that the main in vivo anti-tumor target of CTD is protein phosphatases 2A (PP2A) [9,[10][11][12][13] , suggesting that PP2A inhibition may be the underlying anti-tumor mechanism for CTD. However, as PP2A is generally considered a tumor suppressor, inhibiting PP2A should promote, not suppress tumor growth. Yet, it is also clear that PP2A is involved in many signaling pathways and could act in distinct fashions. A PP2A-inhibiting compound could potentially bind more favorably to a certain subtype or subtypes of PP2A, and/or exhibits organ/tissue specific accumulation, therefore exerting a growth inhibitory effect on specific types of tumors. Thus, inhibiting PP2A by CTD and other PP2A specific drugs remains a possible strategy for achieving antitumor therapeutic benefit. It follows that CTD could be uniquely effective for a subset of liver cancers and/or gastro-esophageal cancers. Accordingly, therapies based on CTD and/or its functional derivatives remain a promising strategy when effective treatments for liver cancers is lacking.
Meanwhile, given the extraordinary toxicity of CTD [4,5] , a major effort has been focused on developing CTD derivatives with a better toxicity profile. This has led to the development of Norcantharidin (NCTD), the demethylated derivative of CTD [6][7][8] . It was originally assumed that CTD and NCTD have similar modes of action and hence exert their anti-cancer activities through shared mechanism(s), i.e. by inhibiting PP2A [14][15][16][17] . However, in order to develop these compounds as anticancer treatments for liver cancers, it is important to empirically determine the modes of action of CTD and NCTD.
In this study, we set out to define the IC50s of CTD and NCTD in a large panel of human cancer cell lines representing twelve different types of human cancers to determine the susceptibility of specific cell lines and/or cancer types to the growth inhibitory effect of CTD, and to determine whether CTD and NCTD affect cells similarly or in distinct fashions.

Cell lines and culture condition
The human cancer cell lines NCI60 were from ATCC. The gastric cancer cell lines and the hepatocellular carcinoma cancer cell lines were gifts from Dr. Youyong Lu of Beijing Institute for Cancer Research (Beijing, China) and Dr. Guangbiao Zhou of Institute of Zoology, Chinese Academy of Sciences (Beijing, China). The cholangiocarcinoma cell lines QBC939, HCCC9810 and RBE were from the Procell Life Science &Technology (Wuhan, China). All cells were maintained in RPMI-1640 medium (Gibco, United States) supplemented with 10% fetal bovine serum (Gibco, United States) and incubated at 37℃ in a humidified atmosphere of 5% CO2 in air.

Cell growth inhibition assay
IncuCyte ZOOM Live-Cell Imaging system (Essen Bioscience, United States) was used to monitor the kinetics of cell growth inhibition by CTD or NCTD (MANSTINE, China) in 72 human cancer cell lines. Cells were seeded at 3,000 cells/well in 96-well plates and treated with increasing concentrations of CTD (0-32 μM) or NCTD (0-160 μM) in RPMI1640 with 10% FBS. The plate was scanned and phase-contrast images were acquired in real time every 4 hours from 0 to 72 hours post treatment. Ratios of cell growth confluence in CTD or NCTD treated cells compare to vehicle were plotted in Microsoft Excel. The growth curves for individual cancer lines were generated using GraphPad Prism v.5 software. IC50 and IC90 values were calculated by logistic regression with Probit Analysis using SPSS program.

Clonogenic survival assay
Cells were trypsinized into single-cell suspension and seeded at 200 cells/well in 6-well plates and treated with increasing concentrations of CTD (0-5 μM) and incubated for 10 d to allow for colony growth. Then, the cells were fixed and stained with crystal violet, and colonies having at least 50 cells were counted under a microscope.

DNA content-based flow assay
Flow cytometric analysis was performed to identify DNA content and to measure the percentage of SubG1 cells after PI staining in PBS as described. Briefly, CTD or NCTD treated cells were suspended and fixed at 75% alcohol overnight. Then, the cells were stained with 50 µM propidium iodide and incubated for 15 min at room temperature and analyzed by a flow cytometer (BD, USA). The percentage of apoptotic cells with a DNA content of less than that of G1 cells was estimated with Listmode software. In addition, cell cycle analysis was performed simultaneously with Multicycle software.

Statistical analysis
Data are presented as ± SD, and statistical analyses were performed using one-way ANOVA for multiple group comparisons. P < 0.05 was considered statistically significant. Mean values and SD were calculated for experiments performed in triplicate.

Results
Cell lines of liver and gastric cancers are highly sensitive to CTD.
In order to determine whether CTD exhibits any selectivity toward abdominal cancers, we decided to assess and compare the relative sensitivities of 72 cell lines that represent 12 major types of human cancers to the growth inhibitory effect of CTD. Traditionally, such an experiment would involve the use of the NCI60 panel of cell lines that represent the 9 major cancer types of the western society. In this case, however, additional lines representing gastric and liver cancers were also included, because these two types of cancers had been claimed to benefit from treatments with CTD, which was first isolated from Mylabris. The use of the semi-high-throughput IncuCyte system has enabled efficient and accurate assessment of the growth inhibitory effects of CTD on a large number of cell lines. Moreover, the data output has allowed case-by-case examination of the data set and accurate computation of the IC50 value for each tested cell lines. Thus, the IC50s for a total of 72 cell lines representing 12 major types of human cancer were established (Table 1, 2). Based on the mean values of the calculated IC50s, it is clear that cell lines from several types of cancers, including those of the prostate cancers, gastric cancers, CCs and hepatoma all have a mean IC50 value below 5 μM (Table 3). Of note, although the mean IC50 value for all of the HCC cell lines is not the lowest, a careful examination of the data set reveals that a subset of these cell lines is among those with the lowest IC50 values (Fig. 1, Table  2).  Clonogenic survival assay was then performed on selected cell lines to further verify the validity of the data obtained using the IncuCyte system. For this experiment, HepG2 and QBC939 were chosen to represent those that were most sensitive to CTD while EKVX the most resistant. The result indicates that HepG2 and QBC939 are indeed highly sensitive to the growth inhibitory effect of CTD, while EKVX remained largely unaffected even at the highest dose examined (Fig. 2). Thus, these data demonstrate that cell lines of different types of human cancers exhibited different sensitivities toward the growth inhibitory effect of CTD. In particular, all gastric cancer cell lines and CC cell lines and a subset of HCC cell lines are among the most sensitive ones to the growth inhibitory effect of CTD.

Individual types of cancer cell lines exhibit distinctive trends of sensitivity toward CTD and NCTD.
When the values of IC90 of CTD and NCTD in each cancer type were compared, it became apparent that for the most past, there is not a tight correlation between the two rankings. For example, for the HCC cell lines, the cells lines with the lowest and the highest rank for CTD and NCTD were Hep3B and SMMC7721; and SNU-449 and Hep3B, respectively (Table 4). A similar discordance between the rankings of IC90 was also observed in other cell lines (Table 5). Thus, there is clearly a discordance in terms of the rankings of sensitivities toward CTD and NCTD among multiple subgroups of cancer cells lines. Such a discordance among multiple subgroups of cancer cell lines between the two compounds, therefore, argues against the notion that the two compounds exert their growth inhibitory effects via similar mechanisms.

CTD and NCTD inhibit growth of HepG2 cells via different mechanisms.
The clear discordance between the growth inhibitory effects of CTD and NCTD within cell lines of individual cancer types prompted us to examine whether these two drugs might in fact cause their respective growth inhibitory effects through different mechanisms. Growth inhibition can be the result of increased cell death, decreased cell cycle progression, or both. Thus, DNA content-based flow cytometry analysis was used to determine the cell cycle progression and/or cell death of HepG2 cells following the treatments with CTD or NCTD. Indeed, when treated at equally potent growth inhibitory concentrations of CTD (IC50, 3 μM; IC90, 5 μM) and NCTD (IC50, 18 μM, IC90, 55 μM), only NCTD, but not CTD caused a significant increase in the sub-G1 fraction, indicative of increased apoptosis; while CTD caused a G1 cell cycle arrest (Fig. 3). These data indicate that while a cytostatic effect is the primary cause of growth inhibitory effect of CTD, cytotoxic effect is the main contributing factor for the growth inhibitory effect of NCTD.

Fig. 2. Clonogenic survival data of the three representative cell lines.
HepG2 and QBC939 were used to represent those that were most sensitive to CTD while EKVX the most resistant. The result showed that HepG2 and QBC939 were indeed highly sensitive to the growth inhibitory effect of CTD (P < 0.01), while EKVX remained resistant to the treatment the highest dose examined. Data are expressed as means ± SD.

Discussion
Despite the long-standing claim that CTD and/or NCTD have clinical indication for other abdominal cancers, in particular liver cancers, a systemic comparison among the various types of human cancer cell lines have not been performed. One possible reason for the lack of such comparison is that the gold-standard cell panel that is usually used for such an experiment, the NCI60 panel, does not include cell lines of liver cancers and gastric cancers [20] . The experimental data presented here indicate that a significant fraction of cell lines derived from liver cancers including HCCs and CCs, are among the most sensitive cell lines toward the growth inhibitory effect of CTD, lending support for the ancient claim that CTD and Mylabris were effective anticancer agents for liver cancers [5] . Interestingly, other types of GI cancers, including gastric and colon cancers, do not appear to be particularly more sensitive to CTD. Thus, despite the fact that CTD is a potent poison, it remains feasible to achieve therapeutic anti-cancer benefit for liver cancers with the tolerable doses of CTD. Accordingly, additional effort in finding the more effective use of CTD and its derivatives for anti-liver cancer treatment is warranted.
Intriguingly, however, we found that there existed a wide spread discordance regarding the growth inhibitory potency between CTD and NCTD toward individual subgroups of cancer cell lines. This finding is in stark contrast to the current assumption that CTD and NCTD exerts its anti-cancer effect via similar mechanism(s) [4] . Rather, it has raised the possibility that these two compounds likely exert their growth inhibitory effects and hence anti-tumor effects through different mechanisms, perhaps by acting upon distinct sets of molecular targets [9][10][11] . Indeed, we showed here that CTD and NCTD caused the growth inhibitory effect of HepG2 cells with different mechanisms. Interestingly, more recent structurefunction studies have revealed that the two methyl group that are present in CTD but not in NCTD play an important role in enhancing the binding affinity toward PP2A [19] . It follows, therefore, that NCTD, a demethylated derivative of CTD, might have different target specificities than CTD. This then raised the possibility that in vivo, CTD and NCTD may also exert their antitumor effects through different mechanisms.
Taken together, the new information presented in this report have called into question the notion that NCTD is a true functional derivative of CTD, the original entity that had been claimed to have potent antitumor effect against abdominal tumors, particularly liver cancers. Accordingly, it will be important to clearly separate these two entities in terms of their antitumor effect as well as mechanisms in order to make better use of these agents and their derivatives for anticancer treatments.
Over all, the data presented here lend further support to the ancient claim that Mylabrsis had unique indication for abdominal masses, particularly liver cancers. Thus, CTD-based regimen(s) remain a promising option for liver cancers including perhaps both HCCs and CCs. However, it is imperative to clearly separate CTD and NCTD. Specifically, although NCTD remains a promising lead for developing effective antitumor agent, it is not a good representative of CTD. Furthermore, for liver cancers, the focus should be on CTD, not its demethylated derivative NCTD and the derivatives thereof. Accordingly, targeted inhibition of PP2A in the liver or within the liver cancer microenvironment should be a main objective.


CHEMISTRY OF CORDYCEPS SINENSIS: A WONDER DRUG FOR ACCLIMATIZATION PURPOSE IN UNREACHED HIGH ALTITUDE AREAS OF HIMALAYAN HILLS

High altitude area of Himalayan hills along Indo-Tibetan border especially 11000-14000 ft above Mean Sea Level is considered to be unreached and unexplored due to adverse climatic conditions. Cordyceps sinensis (Berk.)Sacc. is much valued traditional medicine of this area because of its prominent nutraceutical and medicinal properties. Cordycepin (up to 120 ppm) is the bioactive component of this mushroom. Its utilization for performance enhancement and human acclimatization in the high altitude by curing hypoxia conditions is well established. Hence, nutritional supplements containing C. sinensis may be beneficial for local inhabitants and our Armed Forces deployed in unreached high altitude regions.

and it fetches the price of INR 10 -15 Lakh/kg. C. sinensis has been overexploited in the last three decade, and is bearing the ecological pressure of extinction from ecosystem, which is a very dangerous alarming situation for this species.
Worldwide the efforts of in vitro laboratory culture of this fungus have been undergone to cater the needs of human consumption, and for survival of this species under its natural habitat. Owing to high potency of performance enhancing activity of C. sinensis, Defence Institute of Bio-Energy research (DIBER) has already developed the protocol for in-vitro mycelial culture of this mushroom, and this technology has been transferred to the industries for human welfare. Based on morphology, genetics and biochemical assessment, this in vitro cultured mycelium has been reported very similar to natural samples of C. sinensis.
Cordyceps has been assessed for its chemical composition and bioactive ingredients. Cordycepin (3"deoxyadenosine), adenosine and N-6-(2-hydroxyethyl) adenosine have been identified as quality indexing ingredients of Cordyceps. Cordycepin is a principal bioactive compound of C. sinensis; it is an important nucleoside analog and secondary metabolite of extra-cellular origin. The observation reveals that a little variation in the nutritional components is observed in different wild samples of Cordyceps, but there is a distinct variation in the secondary metabolites in natural samples as well as in cultivated samples. A considerable difference in the concentration of Cordycepin and other bioactive molecules was observed in different culture methodologies, which are having modifications in culture parameters such as light, temperature, Oxygen concentration and substrate used.
II. BIOCHEMICAL COMPOSITION C. sinensis samples differ significantly in chemical composition because of their different geographical origins and culture conditions. In various cases, analytical methods are major reasons for the considerable differences in the biochemical compositions. C. sinensis samples have been studied for their nutraceutical and bioactive constituents.

Nutritional constituents: 2.1.1. Carbohydrates
Carbohydrates are the macromolecules having formula C n (H2O) y , where y ≤ n. carbohydrates are primarily the energy providing bio-molecules required for our daily energy need. Cordyceps sinensis is a rich source of carbohydrates and is composed of monosaccharides, disaccharides, oligosaccharides including cyclofurans, beta-glucans; beta-mannans;, and complex polysaccharides. It contains 43-48% total carbohydrates out of which only 25 -30% have been assessed by anthrone method. Chemically, it contains 40.5% glucose, 35.4% mannose and 14% galactose units. It contains 26 -29% of polysaccharides. In the fungal kingdom, and particularly in Cordyceps spp., polysaccharides are the best known and understood medicinally active compounds. 7 Polysaccharides effectively regulate blood sugar and facilitate antimetastatic and anti-tumor effects. 8

Proteins
Proteins are the second next abundant biomolecule after carbohydrates which are required for body building and body maintenance. C. sinensis contains 22 -28% crude proteins. It is having almost 12 amino acids including 8 essential amino acids, which are necessary for human body. Essential amino acids cannot be synthesized by the body but are necessary for good health. Amino acids are essential for developing the muscles, tendons, organs, glands, nails and hair. Growth, repair and maintenance of all cells are dependent upon amino acids. 9

Fat and lipid
C. sinensis is having 4.8-6.5% fat content consisting of twenty-eight saturated and unsaturated fatty acids and their derivatives. There are two major classes of unsaturated fatty acids -Omega-6 & Omega-3 class and Omega-9 class. Fatty acids regulate blood pressure, cholesterol and triglyceride levels, and are having a positive impact on cardiovascular system. 10

Crude fibers
C. sinensis being as a mushroom contains 6.8-10.5% crude fabric. Fibres are very important for digestive tract as they increase the surface area of food and make the digestion process easier. 11

Vitamins
C. sinensis contains vitamin B-1, B-2, B-12, and E. Vitamin B-1 is essential for proper functioning of central and peripheral nervous system. Vitamin B-2 is essential for energy metabolism, cell formation and growth. Vitamin B-12 is essential for carbohydrates, proteins and fats metabolism pathways and it improves the function of nervous system. Vitamin E is essential in keeping the cells of muscle, heart, testes, liver and nerves alive and functioning.

Minerals 12
Various macro and micronutrients are present in C. sinensis which are adding the value to its nutritional benefits. Mineral contents of C. sinensis are discussed below.

 Potassium
Potassium is important in maintaining fluid and electrolyte balance in the body. Potassium regulates heart condition and supports osmotic pressure. C. sinensis contains 4.75 -6.30 mg/g of potassium contents.

 Sodium
Sodium is an important element for mineral metabolism. Sodium maintains electric potential in tissues. It is essential for osmotic pressure regulation and proper nerve functioning. C. sinensis contains 2.15 -3.20 mg/g of sodium contents.

 Calcium
Calcium is a fundamental material used in mineralization of bones, and its presence is essential for proper functioning of muscles and nerves. C. sinensis contains 360-520 ppm of calcium contents.

 Magnesium
Magnesium is an essential nutritional component for a healthy diet. C. sinensis contains 210-240 ppm of magnesium contents. Over 300 biochemical reactions in the body are regularized by magnesium. Magnesium maintains regular heart rhythm, supports a healthy immune system and keeps bones strong.

 Iron
Iron is involved in oxygen transport and regulation of cell growth and differentiation. C. sinensis contains 14.6 -16.2 ppm of iron contents. A deficiency in iron limits oxygen delivery to cells, resulting in fatigue and poor work performance.

 Copper
Copper is an essential microelement for the health of all living organisms. C. sinensis contains 3.0-4.0 ppm of copper contents. Copper plays an important role in facilitating iron uptake in human body.

 Manganese
Manganese acts as a co-enzyme to assist metabolic progression in the human body. C. sinensis contains 2.0 -3.0 ppm of manganese contents Manganese helps to maintain healthy bone structure and is involved in building essential enzymes for developing bones. Manganese is also actively involved in forming connective tissues, absorption of calcium, proper functioning of the thyroid and regulation of blood sugar levels.

 Zinc
Zinc is an essential trace element for cell growth and division and to strengthen immune system. Zinc accelerates the renewal of the skin cells and is essential for maintaining healthy vision. C. sinensis contains 11.6 -18.4 ppm of zinc contents.

Bioactive ingredients 2.2.1. Nucleosides
C. sinensis contains various types of nucleosides including adenosine, 2-deoxyadenosine, 3deoxyadenosine (cordycepin), guanosine and N-6-(2hydroxyethyl) adenosine. These nucleosides are very beneficial and play important role in the regulation of various physiological processes in the central nervous system, if consumed as nutraceuticals. They have great participation in DNA and RNA synthesis. The energy regulation of human body is dependent on phosphorylation process of these nucleosides. Cordycepin and adenosine has been identified as major bioactive components of Cordyceps spp. 13

Cordycepin
Cordycepin or 3"-deoxyadenosine is a major bioactive component of C. sinensis. It is produced by this mushroom in hypoxia conditions. Cordycepin is the extracellular secondary metabolite of C. sinensis, which is produced under stressed environmental conditions in high altitude regions. Initially, this molecule was isolated from C. militaris. Characterization of this molecule was carried out by Chen and Chu using nuclear magnetic resonance (NMR) and infrared spectroscopy (IR) in an extract of C. sinensis. 14 The structure of cordycepin is as under. (Fig. 1)

Fig.1 Molecular structure of Cordycepin or 3'deoxyadenosine
There is a lack of oxygen on C-3 in ribose sugar and hence it is an excellent terminator of transcription process. When Cordycepin is introduced to bacterial cell undergoing DNA replication, it produces a defective DNA that triggers bacteria cell to undergo apoptosis (programmed cell death). It also inhibits the m-RNA formation in some bacteria. Cordycepin is very effective against all sorts of bacteria that have developed resistance to the other antibiotics. It also shows anti-viral properties due to 3-deoxy positions. The best known medicinal action of Cordyceps is to increase of physical stamina and to cure hypoxia conditions at high altitude regions. Cordyceps improves the internal balance mechanism, thus making the utilization of oxygen more efficient. By curing the hypoxia conditions it helps in acclimatization of humans in unreached high altitude regions. The concentration of cordycepin in different Cordyceps strains differ significantly, even in some cases it is not detectable. In natural samples cordycepin contents may vary from negligible to 120 ppm.

Adenosine
Adenosine is composed of adenine and ribose sugar, and it is a very essential nucleoside for living organisms. It gives energetic nucleotides viz. adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) on phosphorylation. All these nucleotides are intensively involved in metabolism, and regulate the energy requirement of various human physiological processes. 15 further; it is well known that these nucleotides are essential for DNA repair process and m-RNA synthesis. Different strains of C. sinensis show variability in adenosine contents, RP-HPLC chromatogram shows 30-250 ppm adenosine content in different samples. The molecular structure of adenosine is given below (Fig. 2)

Fig.2 Molecular structure of Adenosine 2.2.2. Polyphenols
Phenolic compounds have been proven a major class of phytochemicals, which are accountable for inhibiting the oxidative damage caused by free radicals. 16 C. sinensis contains a variety of polyphenolic compounds including flavonoids, flavonols and proanthocyanidins. Polyphenolics were extracted with the help of water and alcohol. The concentration of phenolic compounds was higher in the alcoholic extracts, when compared with the water extracts. The polyphenol content in C. sinensis is given below. [Fig 3].

Sterols
Various sterols are reported in Cordyceps spp. including Delta-3 ergosterol, ergosterol, ergosterol peroxide, 3-sitosterol and campeasterol. 17 Sterols such as campesterol can prevent "bad" cholesterol from being absorbed into the bloodstream. Instead of clogging up arteries and impairing artery function, the cholesterol is eliminated as waste with the help of these sterols. In this manner, sterols in the C. sinensis may reduce the risk of coronary heart disease.

Cordycepic acid
Cordycepic acid or D-mannitol is well known polyhydroxy alcohol present in C. sinensis, It is an active component of C. sinensis. C. sinensis has 6-8% of cordycepic acid. In international market quality of C. sinensis and its culture is measured in terms of cordycepic acid content besides the nucleoside analogues. 18 It has diuretic action and is used as a prophylaxis against postoperative acute renal failure. The molecular structure of cordycepic acid or D-mannitol is given below.

Fig.4 Molecular structure of cordycepic acid
III. CONCLUSION C. sinensis grows in high altitude regions especially 11000-14000 ft above mean sea level of western Himalayan region. It is very well known for its beneficial effects in the high altitude regions especially performance enhancing capabilities and curing hypoxia conditions. Local inhabitants and our soldiers deployed in high altitude regions are much desirous to get support for their acclimatization under this challenging situation. The chemical study of C. sinensis is definitely a great effort to support nutraceutical industries in developing pharmaceuticals and nutraceuticals for our local inhabitants and soldiers. This will be the way, by which they will be able to face adverse climatic conditions in efficient manner in unreached Himalayan locations. From this study, it is evident that C. sinensis has good nutritional value and it contains variety of chemical constituents like cordycepic acid, polyphenols, polysaccharides, sterols and free amino acids. Nucleosides like cordycepin and adenosine are the potent bioactive molecules in C. sinensis. Besides the performance enhancing capabilities and curing hypoxia conditions cordycepin has been proven to be effective against a variety of bacterial and viral infections. Thus, in future more R&D efforts are required for developing nutraceuticals and pharmaceuticals from C. sinensis to support local inhabitants and our Armed Forces deployed in far reaches of high altitude regions to protect our international boundaries along Indo-Nepal and Indo-Tibatan borders.