Ginsenoside Rb1 alleviates 3-MCPD-induced renal cell pyroptosis by activating mitophagy.

Ginsenoside Rb1 (Gs-Rb1) is among the most significant effective pharmacological components in ginseng. 3-monochloropropane-1,2-diol (3-MCPD), a chloropropanol-like contaminant, is produced in the production of refined oils and thermal processing of food. Pyroptosis is a type of programmed cell death triggered by inflammasomes. Excessive pyroptosis causes kidney injury and inflammation. Previous studies have revealed that 3-MCPD induced pyroptosis in mice and NRK-52E cells. In the present study, we find that Gs-Rb1 attenuates 3-MCPD-induced renal cell pyroptosis by assaying GSDMD-N, caspase-1, IL-18, and IL-1ß in mice and NRK-52E cells. In further mechanistic studies, we show that Gs-Rb1 removes damaged mitochondria via mitophagy and reduces intracellular reactive oxygen species (ROS) generation, therefore alleviating 3-MCPD-induced NOD-like receptor family pyrin domain containing 3 (NLRP3) activation and pyroptosis. The above results are further validated by the addition of autophagy inhibitor Chloroquine (CQ) and mitophagy inhibitor Cyclosporin A (CsA). Afterward, we explore how Gs-Rb1 activated mitophagy in vitro. We determine that Gs-Rb1 enhances the protein expression and nuclear translocation of Transcription factor EB (TFEB). However, silencing of the TFEB gene by small interfering RNA technology reverses the role of Gs-Rb1 in activating mitophagy. Therefore, we conclude that 3-MCPD damages mitochondria and leads to ROS accumulation, which causes NLRP3 activation and pyroptosis in ICR mice and NRK-52E cells, while Gs-Rb1 mitigates this phenomenon via the TFEB-mitophagy pathway. Our findings may provide new insights for understanding the molecular mechanisms by which Gs-Rb1 mitigates renal injury.

Identification of genes showing altered DNA methylation and gene expression in the renal proximal tubular cells of rats treated with ochratoxin A for 13 weeks.

Ochratoxin A (OTA) is a mycotoxin that causes renal carcinogenicity following the induction of karyomegaly in proximal tubular cells after repeated administration to rats. Here, we performed gene profiling regarding altered DNA methylation and gene expression in the renal tubules focusing on the mechanism of OTA-induced carcinogenesis. For this purpose, OTA or 3-chloro-1,2-propanediol (3-MCPD), a renal carcinogen not inducing karyomegaly, was administered to rats for 13 weeks, and DNA methylation array and RNA sequencing analyses were performed on proximal tubular cells. Genes for which OTA altered the methylation status and gene expression level, after excluding genes showing similar expression changes by 3-MCPD, were subjected to confirmation analysis of the transcript level by real-time reverse-transcription PCR. Gene Ontology (GO)-based functional annotation analysis of validated genes revealed a cluster of hypermethylated and downregulated genes enriched under the GO term "mitochondrion," such as those associated with metabolic reprogramming in carcinogenic process (Clpx, Mrpl54, Mrps34, and Slc25a23). GO terms enriched for hypomethylated and upregulated genes included "response to arsenic-containing substance," represented by Cdkn1a involved in cell cycle arrest, and "positive regulation of IL-17 production," represented by Osm potentiating cell proliferation promotion. Other genes that did not cluster under any GO term included Lrrc14 involved in NF-κB-mediated inflammation, Gen1 linked to DNA repair, Has1 related to chromosomal aberration, and Anxa3 involved in tumor development and progression. In conclusion, a variety of genes engaged in carcinogenic processes were obtained by epigenetic gene profiling in rat renal tubular cells specific to OTA treatment for 13 weeks.

Activating autophagy and ferroptosis of 3­Chloropropane­1,2­diol induces injury of human umbilical vein endothelial cells via AMPK/mTOR/ULK1.

3­Chloropropane­1,2­diol (3­MCPD) is an internationally recognized food pollutant. 3­MCPD has reproductive, renal and neurotoxic properties. However, whether 3­MCPD induces human umbilical vein endothelial cell (HUVEC) injury has not been previously reported. In the present study, HUVECs were treated using 2 µg/ml 3­MCPD for 24 h at 37˚C. The effects of 3­MCPD on HUVEC proliferation and cell cycle arrest, death and senescence were then assessed using Cell Counting Kit­8 (CCK­8), flow cytometry and ß­galactosidase staining, respectively. Whether 3­MCPD induced ferroptosis was evaluated using JC­1 and FerroOrange staining and transmission electron microscopy. A small interfering RNA targeting AMPK was used to assess whether 3­MCPD promoted ferroptosis via AMPK signaling. The results demonstrated that 3­MCPD inhibited HUVEC proliferation in a dose­dependent manner and induced cell cycle arrest. Furthermore, 3­MCPD promoted senescence in HUVECs with elevated DNA damage and cell death. The CCK­8 results demonstrated that ferroptosis and autophagy inhibitors significantly reversed cell death caused by 3­MCPD. Moreover, 3­MCPD increased mitochondrial membrane potential, which indicated that 3­MCPD contributed to mitochondrial dysfunction. 3­MCPD also markedly increased intracellular Fe2+ levels and lipid peroxidation in HUVECs. The present study assessed the underlying mechanism by which 3­MCPD activated autophagy and ferroptosis in HUVECs. The data demonstrated that 3­MCPD significantly increased phosphorylation levels of AMPK and unc­51 like autophagy activating kinase (ULK1) but significantly decreased phosphorylation of mTOR in HUVECs. Furthermore, silencing of AMPK significantly reversed the increase in autophagy, lipid peroxidation and Fe2+ induced by 3­MCPD. In conclusion, 3­MCPD demonstrated a significant damaging effect on HUVECs via induction of autophagy and ferroptosis; such effects may be mediated by AMPK/mTOR/ULK1 signaling. To the best of our knowledge, the present study was the first to demonstrate the mechanism of 3­MCPD­induced vascular endothelial cell injury and lays a molecular foundation for the prevention of 3­MCPD­related vascular diseases.

1,3-dichloro-2-propanol induced hepatic lipid accumulation by inhibiting autophagy via AKT/mTOR/FOXO1 pathway in mice.

Our study investigated the effects of food contaminant 1,3-dichloro-2-propanol (1,3-DCP) on hepatic lipid metabolism and its mechanism. We found that triglyceride (TG), total cholesterol (TC) and the number of lipid droplets (LDs) were increased in the liver of C57BL/6 mice given intragastric administration of 1,3-DCP for 30 days. Meanwhile, 1,3-DCP inhibited autophagosomes and lysosomes formation, reflected by decreased LC3-II, LAMP1, LAMP2, CTSD, CTSB expression, increased p62 expression and decreased LC3 fluorescence. Subsequently, we detected the changes of hepatic lipid accumulation caused by 1,3-DCP using an autophagy inducer or inhibitor. In vivo, Hepatic lipid accumulation caused by 1,3-DCP was mitigated by the autophagy inducer Rapa. On the contrary, the autophagy inhibitor (chloroquine or 3-methyladenine) further exacerbated hepatic lipid accumulation caused by 1,3-DCP. 1,3-DCP reduced the number of autophagosomes encapsulated LDs, assessed by colocalization of LD and LC3. These data demonstrated that 1,3-DCP induced lipid accumulation by inhibiting autophagy. We further investigated the mechanism of 1,3-DCP-inhibited autophagy and found 1,3-DCP increased the ratios of p-AKT/AKT, p-mTOR/mTOR, p-FOXO1/FOXO1, decreased FOXO1 nuclear localization in vivo. These proteins may be involved in the regulation of 1,3-DCP-mediated autophagy. We detected the changes in autophagy marker protein LC3-II and lipid accumulation using an AKT inhibitor ARQ-092 or a mTOR inhibitor rapamycin in HepG2 cells. Compared with 1,3-DCP group, lipid accumulation was decreased, LC3-II and FOXO1 nuclear localization were increased, p-FOXO1 levels were decreased in HepG2 cells pretreated with ARQ-092 or rapamycin. Taken together, these data revealed that the effects of 1,3-DCP on lipid accumulation by inhibiting autophagy were dependent on AKT/mTOR/FOXO1 signaling pathway. Our study not only supplied the mechanism of 1,3-DCP toxicity, but also provided experimental basis for effective intervention measures of 1,3-DCP toxicity.

LKB1/AMPKα signaling pathway and mitochondrial fission/fusion dynamics regulate apoptosis induced by 3-chlorpropane-1,2-diol in HEK293 cells.

Mitochondrial dynamics and bioenergetics are considered play pivotal roles in the maintenance of mitochondrial function and cell viability. During the widely distributed food contaminant 3-chlorpropane-1,2-diol (3-MCPD) induced nephrotoxicity, mitochondrial morphology and function were impaired, but the specific mechanism responsible for the process has not been fully elucidated. In the present study, using an in vitro human embryonic kidney 293 (HEK293) cell culture model, the role of LKB1/AMPK pathway and mitochondrial fission and fusion dynamics in 3-MCPD-induced cell apoptosis was investigated by using the AMPK inhibitor dorsomorphin and mitochondrial division inhibitor 1 (Mdivi-1), respectively. The results revealed that 3-MCPD significantly decreased the ATP levels, activated the energy-sensing regulator AMPKα and its upstream protein kinase LKB1, disrupted mitochondrial dynamics equilibrium characterized by promoting division and inhibiting fusion, thus inducing cell apoptosis. Notably, suppression of AMPK by dorsomorphin mitigated 3-MCPD-induced cytotoxicity through improvement of the function and dynamics of mitochondria and alleviated apoptosis via the mitochondria-dependent pathway. Moreover, inhibition of mitochondrial fission by Mdivi-1 protected against apoptosis induced by 3-MCPD. Taken together, these results suggest that 3-MCPD triggers apoptosis through activation of LKB1/AMPKα signaling pathway and regulation of mitochondrial fission and fusion dynamics in HEK293 cells.

3-MCPD (3-monochloro-1,2-propanediol) inhibit myogenic differentiation in murine skeletal myoblasts.

3-Monochloro-1,2-propanediol (3-MCPD) is a chemical compound that is unintentionally produced during food processing such as acid hydrolysis. There has been reports regarding the role of this chemical compound in reproductive toxicity, as well as genotoxicity, neurotoxicity, and kidney toxicity. In this study, the in vitro muscle toxicity of 3-MCPD was assessed using C2C12 myoblast cells. The reduction in muscle regulatory factors (MRFs), which is related to muscle differentiation, was identified as significant with the increase concentration of 3-MCPD. Also, significantly decreased protein expression in mTOR and p70S6 kinase, which are the downstream targets of the pathway associated with muscle synthesis, was also confirmed. Therefore, the inhibitory effect of 3-MCPD on muscle differentiation is considered to be the cause of suppressing mTOR and p70S6 kinase expression. In conclusion, it was confirmed that 3-MCPD inhibits muscle differentiation in C2C12 myoblasts through suppressing the expression of several genetic factors involving muscle differentiation.

3-Chloro-1, 2-propanediol inhibits autophagic flux by impairment of lysosomal function in HepG2 cells.

3-chloro-1, 2-propanediol (3-MCPD) is a well-known contaminant that was produced in the thermal processing of food. Dietary intake represents the greatest source of exposure to 3-MCPD. Autophagy is an important catabolic pathway that plays an important role in liver physiological function. Evidence suggests that 3-MCPD exposure causes toxicity in liver, but the mechanism remains unknown. Here, we explored the effects of 3-MCPD on autophagic flux and traced the molecular mechanism in HepG2 cells. The data showed 3-MCPD exposure promoted the accumulation of autophagosomes in HepG2 cells. Subsequently, by detected te expression of LC3-Ⅱ and P62 and transfection of mRFP-GFP-LC3 adenovirus, we found that the accumulation of autophagosomes was caused by inhibition of autophagic flux. After that, we investigate lysosomal function and found that 3-MCPD induced lysosomal alkalinization. Further, we detected the expression of TFEB, which is a key nuclear transcription factor in control of lysosome biogenesis and function. We found that 3-MCPD inhibited the nuclear expression of TFEB and mRNA levels of some target genes of TFEB. In order to further verify the role of TFEB in autophagic flux blockage in HepG2 cells induced by 3-MCPD, we overexpressed TFEB by transfection with adenovirus and found that both autophagy inhibition and lysosomal alkalization induced by 3-MCPD were alleviated. These results suggested that 3-MCPD could induce the autophagic flux blockage in HepG2 cells. The possible mechanism was due to the destruction of lysosomal function.

3-Monochloropropane-1,2-diol (alpha-chlorohydrin) disrupts spermatogenesis and causes spermatotoxicity in males of the Egyptian fruit-bat (Rousettus aegyptiacus).

We evaluated the sterilizing effect of 3-monochloropropane-1, 2-diol (3-MCPD) in male Egyptian fruit bats (Rousettus aegyptiacus). We used three groups. One was treated with 70 mg/kg 3-MCPD for 4 days. The second group was treated with 3-MCPD as a bait formulation (known concentration of 3-MCPD mixed with a known amount of food). The third group was untreated controls. We compared the weights of the reproductive organs, histology of the testes, occurrence of spermatogenesis, and the count, motility and abnormalities of epididymal sperm of treated males with those of the untreated control group. 3-MCPD caused significantly decreased weights of reproductive organs, several testicular histological alterations and spermatogenic arrest accompanied by significant decreases in sperm count and motility, and significantly increased number of abnormal sperm. 3-MCPD bait was readily accepted by the animals. 3-MCPD, even in low doses and after limited exposure, disrupted spermatogenesis in males of the Egyptian fruit bat. Our findings have potential value for public health and agricultural authorities, and for vertebrate pest managers. 3-MCPD may have application for control of this pest.

Alliin attenuates 1, 3-dichloro-2-propanol-induced lipogenesis in HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway.

Accumulating evidence reveals the association of 1, 3-dichloro-2-propanol (1, 3-DCP) exposure and lipogenesis. Alliin, the most abundant sulphur compound in garlic, has been demonstrated to exhibit hypoglycemic, antioxidant and anti-inflammatory activities. Here, we showed that alliin attenuated lipogenesis induced by 1,3-DCP and that the reduction was due to activation of the AMPK pathway. HepG2 cells exposed to 1,3-DCP exhibited significant increase of triglyceride(TG) and total cholesterol(TC), and alliin reduced the accumulation. Most importantly, alliin could up-regulate the phosphorylation of AMPK and down-regulate protein and gene expressions of SREBP-1; FAS; SREBP-2;HMGCR in 1,3-DCP-induced HepG2 cells. The results demonstrated that alliin was effective on attenuating 1,3-DCP-induced lipogenesis via activation of the AMPK-SREBPs signaling pathway in HepG2 cells.

Effects of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites on DNA damage and repair under in vitro conditions.

3-monochloropropane-1,2-diol (3-MCPD) is a food contaminant that occurs during industrial production processes and can be found mainly in fat and salt containing products. 3-MCPD has exhibited mutagenic activity in vitro but not in vivo, however, a genotoxic mechanism for the occurrence of kidney tumors has not so far been excluded. The main pathway of mammalian 3-MCPD metabolism is via the formation of ß--chlorolactatic acid and formation of glycidol has been demonstrated in bacterial metabolism. The aim of this study was to investigate genotoxic and oxidative DNA damaging effects of 3-MCPD and its metabolites, and to provide a better understanding of their roles in DNA repair processes. DNA damage was assessed by alkaline comet assay in target rat kidney epithelial cell lines (NRK-52E) and human embryonic kidney cells (HEK-293). Purine and pyrimidine base damage, H2O2 sensitivity and DNA repair capacity were assessed via modified comet assay. The results revealed in vitro evidence for increased genotoxicity and H2O2 sensitivity. No association was found between oxidative DNA damage and DNA repair capacity with the exception of glycidol treatment at 20 µg/mL. These findings provide further insights into the mechanisms underlying the in vitro genotoxic potential of 3-MCPD and metabolites.

A 4-week study of four 3-monochloropropane-1,2-diol diesters on lipid metabolism in C57BL/6J mice.

3-Monochloropropane-1,2-diol (3-MCPD) esters have been detected in many foods, which have become a new safety issue worldwide. In the study, we investigated the effect of four 3-MCPD diesters (palmitate diester: CDP; stearate diester: CDS; oleate diester: CDO; linoleate diester: CDL) on lipid metabolism in C57BL/6J mice. The results showed that CDP, CDS, CDO and CDL significantly increased the serum TC, LDL-C levels and liver TG, TC levels at dose of 16.5µmol/kg/day. These results indicated that 3-MCPD diesters could potentially cause hyperlipidemia in C57BL/6J mice. Moreover, oil red O staining confirmed fat accumulation in liver induced by 3-MCPD diesters. Our work will provide more information for safety evaluation of 3-MCPD diesters. However, whether free 3-MCPD or free fatty acids or combined action compensates for the hyperlipidemia effects should be elucidated in the future.

Transitory loss of glia and the subsequent modulation in inflammatory cytokines/chemokines regulate paracellular claudin-5 expression in endothelial cells.

Signaling mechanisms involved in regulating blood-brain barrier (BBB) integrity during central nervous system (CNS) inflammation remain unclear. We show that an imbalance between pro-/anti-inflammatory cytokines/chemokines alters claudin-5 expression. In vivo, gliotoxin-induced changes in glial populations and an imbalance between pro-/anti-inflammatory cytokine/chemokine expression occurred as BBB integrity was compromised. The balance was restored as BBB integrity was re-established. In vitro, TNF-α, IL-6, and MCP-1 induced paracellular claudin-5 expression loss. TNF-α- and IL-6- effects were mediated through the PI3K pathway and IL-10 attenuated TNF-α's effect. This study shows that pro-/anti-inflammatory modulators play a critical role in BBB integrity during CNS inflammation.

(S)-α-chlorohydrin inhibits protein tyrosine phosphorylation through blocking cyclic AMP - protein kinase A pathway in spermatozoa.

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5'-triphosphate (ATP) levels, 3'-5'-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.

Evaluating the anti-fertility potential of α-chlorohydrin on testis and spermatozoa in the adult male wild Indian house rat (Rattus rattus).

To examine the effects of α-chlorohydrin on testis and cauda epididymis in the male house rat (Rattus rattus), 24 adult male rats were segregated into two groups. Group I rats were force-fed daily by intragastric intubation with α-chlorohydrin at a single dose of 1.0 mg/100 g body weight/d for 5, 15, and 45 days. Another group was fed with distilled water, which served as the control. The treated male rats were paired with 24 adult proestrus female rats for 5 days after the last oral treatment and fertility was tested. At the end of the experiments, all of the male rats were weighed and killed by cervical dislocation. The right testes were removed, weighed, and processed for ultrastructural changes of spermatozoa from the cauda epididymis and testis under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The seminiferous tubular area, nuclear diameter of the Sertoli and Leydig cells, percentage of spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, spermatozoa, and Sertoli cells in each group were compared morphometrically. Our results showed that the percentages of primary spermatocytes steadily increased from 5 to 15 days, but primary and secondary spermatocytes decreased significantly at 45 days. There was a steady decline in the percentages of spermatozoa and spermatids at all fixation intervals in the treated animals, but the percentages of spermatogonia and Sertoli cells increased significantly at 15 and 45 days. Seminiferous tubular areas, nuclear diameter of Leydig and Sertoli cells, and fertility rates were reduced after 45 days of treatment. SEM and TEM studies revealed severe morphological abnormalities in the spermatozoa, including deglutination of the acrosomal part, loss of head capsules, and fragmentation of tail fibrils. There was an enhanced anti-fertility effect and a lower number of implantation sites in the rats treated for 5 days. Our results validate α-chlorohydrin as a successful anti-fertility agent that prevents spermatogenesis.

Differential susceptibility of astrocytic and neuronal function to 3-chloropropanediol in the rat inferior colliculus.

We have previously shown that systemic administration of S(+)3-chloropropanediol (3-CPD) produces a morphological loss of astrocytes in specific nuclei of the rodent brain that precedes loss of both neurones and endothelial tight junctions. Here, we have evaluated the differential susceptibility of neuronal and astrocytic function to 3-CPD, in order to see if this parallels the morphological selectivity. To do this, we have developed an in vivo method for monitoring astrocyte function over time by giving hourly 20-min bolus challenge exposures to ammonia via an implanted microdialysis probe and measuring the resulting transient increases in the extracellular glutamine : glutamate ratio. These challenge ammonia exposures evoked a stable response for at least 5 h when the probe was implanted in the rat inferior colliculus, but caused no behavioural response or morphological damage. Although 3-CPD produced a rapid and sustained abolition of the ammonia response within 2 h, the field potential response of inferior collicular neurones to sound fell significantly to 75.0 ± 3.9% pre-dose at up to 8 h but then fell markedly, reaching 20.5 ± 3.7% at 2 days. Blood flow in the inferior colliculus also showed only late changes, increasing substantially at 2 days. Astrocyte damage at the EM level was seen from 3 h, followed by loss of astrocytes from 18 h to a minimum of 7 ± 10% control at 3 days. The rapid abolition of the ammonia response suggests that in addition to selective astrocyte death, 3-CPD also produces an earlier impairment of astrocyte function that precedes loss of neuronal function. This initial functional selectivity of 3-CPD provides a potential investigative tool in neurochemical studies of astrocyte-neuronal interactions.

1,3-Dichloro-2-propanol induces apoptosis via both calcium and ROS in mouse melanoma cells.

We demonstrated that the apoptotic cell death of mouse melanoma cells exposed to 1,3- dichloro-2-propanol (DCP) was Ca(2+) dependent. 1,3-DCP inhibited the growth of mouse melanoma cells in a concentration-dependent manner. Furthermore, a terminal nick-end labeling of fragmented DNA (TUNEL) assay demonstrated that 1,3-DCP-induced apoptosis. Moreover, reactive oxygen species and Ca(2+), generated by mouse melanoma cells that were exposed to 1,3-DCP, resulted in the activation of NF-jB and MAPKs. Finally, treatment with BAPTA, an intracellular Ca(2+) chelator, completely inhibited the apoptosis that was induced by 1,3-DCP treatment of B16F10 cells.

1H NMR to investigate metabolism and energy supply in rhesus macaque sperm.

Sperm ATP is derived primarily from either glycolysis or mitochondrial oxidative phosphorylation. In the present studies, (1)H NMR spectroscopy was used to characterize the metabolite profile in primate sperm treated either with alpha-chlorohydrin (ACH), a known inhibitor of sperm glycolysis or pentachlorophenol (PCP), an uncoupler of oxidative phosphorylation. Sperm were collected from monkeys in the fall and spring, washed and incubated with either the media control, ACH (0.5mM) or PCP (50 microM). Using principal components analysis, PC1 scores plot indicated that the greatest level of variance was found between fall and spring samples and not chemical-treated samples. However, PC4 scores plot did show a consistent effect of ACH treatment. From the PC1 loadings plot, metabolites contributing to the seasonal differences were higher levels of formate in the fall and higher levels of carnitine and acetylcarnitine in the spring as well as possible differences in lipoprotein content. The PC4 loadings plot indicated that ACH treatment decreased lactate and ATP consistent with inhibition of glycolysis. Carnitine also was decreased and acetylcarnitine increased although the latter was not statistically significant. With PCP-treated sperm, no difference between control and treated samples could be discerned suggesting either that primate sperm are insensitive to uncoupling agents or that glycolysis played the more important role in maintaining sperm ATP levels. Overall, NMR studies may prove useful in the development of metabolomic markers that signal sperm metabolic impairments and have the potential to provide useful biomarkers for reproductive health.

Sperm mitochondrial integrity is not required for hyperactivated motility, zona binding, or acrosome reaction in the rhesus macaque.

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.

Microvascular P-glycoprotein expression at the blood-brain barrier following focal astrocyte loss and at the fenestrated vasculature of the area postrema.

The multidrug transporter, P-glycoprotein, expressed at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but its relationship to astrocyte expression is unclear. We have studied P-glycoprotein expression in the inferior colliculus after a temporary loss of blood-brain barrier integrity following chemically induced astrocyte loss and at the fenestrated vascular endothelium of the area postrema. Male Fisher F344 rats given 3-chloropropanediol showed astrocyte loss from 12 to 24 h until the lesion was repopulated 8-28 days later. In non-dosed tissue, P-glycoprotein expression was seen the entire length of platelet endothelial cell adhesion molecule immunoreactive vessels. Within 6 h of dosing, a significant (p<0.05) reduction in the total length of P-glycoprotein immunoreactive vasculature was evident. By 48 h, P-glycoprotein immunoreactivity was heavily fragmented. The total length of P-glycoprotein immunoreactive vessels became minimal at 4 days (p<0.001) but was still present in many vessels. From 6 to 28 days, P-glycoprotein immunoreactivity returned across the inferior colliculus, in parallel with astrocytic repopulation of the lesion, and by 28 days resembled that seen in control tissue. The area postrema showed GFAP immunoreactive astrocytes but which made limited contact with the vasculature, while the platelet endothelial cell adhesion molecule immunoreactive vasculature showed no expression of P-glycoprotein. These findings provide evidence supporting a link between GFAP-astrocyte and P-glycoprotein expression in the mature brain vasculature in vivo.

Effect of an inactivator of glyceraldehyde-3-phosphate dehydrogenase, a fortuitous arsenate reductase, on disposition of arsenate in rats.

The environmentally prevalent arsenate (AsV) is reduced in the body to the much more toxic arsenite (AsIII). Recently, we have demonstrated that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reduction of AsV in the presence of glutathione, yet the role of GAPDH in AsV reduction in vivo is unknown. Therefore, we examined the effect of (S)-alpha-cholorhydrin (ACH), which forms a GAPDH-inhibitory metabolite, on the reduction of AsV in rats. These studies confirmed the in vitro role of GAPDH as an AsV reductase, inasmuch as 3 h after administration of ACH (100 or 200 mg/kg, ip) to rats both the cytosolic GAPDH activity and the AsV-reducing activity dramatically fell in the liver, moderately decreased in the kidneys, and remained unchanged in the muscle. Moreover, the AsV-reducing activity closely correlated with the GAPDH activity in the hepatic cytosols of control and ACH-treated rats. Two confounding effects of ACH (i.e., a slight fall in hepatic glutathione levels and a rise in urinary AsV excretion) prompted us to examine its influence on the disposition of injected AsV (50 micromol/kg, iv) in rats with ligated bile duct as well as in rats with ligated bile duct and renal pedicles. These experiments demonstrated that the hepatic retention of AsV significantly increased, and the combined levels of AsV metabolites (i.e., AsIII plus methylated arsenicals) in the liver decreased in response to ACH; however, ACH failed to delay the disappearance of AsV from the blood of rats with blocked excretory routes. Thus, the GAPDH inactivator ACH inhibits AsV reduction by the liver, but not by the whole body, probably because the impaired hepatic reduction is compensated for by hepatic and extrahepatic AsV-reducing mechanisms spared by ACH. It is most likely that ACH inhibits hepatic AsV reduction predominantly by inactivating GAPDH in the liver; however, a slight ACH-induced glutathione depletion may also contribute. While this study seems to support the conclusion that GAPDH in the liver is involved in AsV reduction in rats, confirmation of the in vivo role of GAPDH as an AsV reductase is desirable.