Chlorpyrifos (CPF), a commonly used cholinesterase-inhibiting insecticide, is lethal at much lower doses to young animals than adults. To explain this higher sensitivity in younger animals, we hypothesized that young rats have less chlorpyrifos-oxonase (CPFOase) activity than adults. To test this hypothesis, CPFOase activity was measured in the brain, plasma, and liver of male, postnatal day 4 (PND4) and adult (PND90) Long-Evans rats. CPFOase is biochemically defined as a Ca(2+)-dependent A-esterase that hydrolyzes chlorpyrifos-oxon (CPFO), the active metabolite of CPE. No brain CPFOase activity was detected at either age. Plasma and liver CPFOase activities were markedly lower at PND4 compared to adult: PND4 plasma and liver CPFOase activities were 1/11 and 1/2 the adult plasma and liver activities, respectively. Because the Km of CPFOase activity was high (i.e., 210-380 microM), it was important to determine if this CPFOase activity could hydrolyze physiologically relevant concentrations (i.e., nM to low microM) of CPFO. This was accomplished by comparing the shifts in the tissue acetylcholinesterase (AChE) IC50 for CPFO in the presence or absence of CPFOase activity. One would expect an increase in the "apparent" IC50 if CPFOase hydrolyzes substantial amounts of CPFO during the 30 minutes the tissue is preincubated with the CPFO. In the adult, both plasma and liver AChE apparent IC50 values were higher in the presence of CPFOase activity, suggesting that the CPFOase in those tissues was capable of hydrolyzing physiologically relevant concentrations of CPFO within 30 minutes. In young animals, however, there was less of a shift in the IC50 curves compared to the adult, confirming that the young animal has less capacity than the adult to detoxify physiologically relevant concentrations of CPFO via CPFOase.
Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one(t-PBO), delta 5-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage.
Aliesterases (carboxylesterases) are serine esterases that can serve a protective role for the target acetylcholinesterase (AChE) during organophosphorus insecticide intoxication because the former esterases are alternate phosphorylation sites. The levels of aliesterase activity in liver and plasma and AChE activity in brain regions were investigated after the intravenous administration of paraoxon (P = O) into female rats. The rats were pretreated intraperitoneally with beta-napthoflavone (BNF), which decreases hepatic aliesterase activity following a 3 day in vivo treatment, and/or tri-o-tolyl phosphate (TOTP) to inhibit aliesterases. The liver aliesterases were inhibited less by P = O in BNF-treated rats than in control rats, which suggests that either BNF exposure may have resulted in aliesterases that are less sensitive to P = O inhibition or BNF may have altered P = O's availability. The BNF treatment did not seem to alter the degree of inhibition of the brain AChE activity following the low dosage of paraoxon (0.04 mg/kg). However, the brain AChE activity in the P = O/TOTP/BNF-treated rats was lower than that in the P = O/TOTP-treated rats, suggesting that BNF also caused changes in systems affecting the disposition of P = O in addition to the changes in the hepatic aliesterases. At the high dosage of paraoxon (0.12 mg/kg), the AChE and aliesterase activities showed a pattern similar to that of the low dosage. This suggests that the aliesterases, as altered by BNF exposure, even when nearly completely inhibited, did not alter the response of the target enzyme, AChE, and, therefore, the magnitude of the toxic response.
Two gold compounds, gold sodium thiomalate (AuTM) and auranofin, are presently in clinical use in therapy of rheumatoid arthritis. In these studies, AuTM administered to Sprague-Dawley rats and three strains of mice, Swiss-Webster, C3H/HeJ, and DBA/2J, were studied with regard to its effect on liver and renal monooxygenases, metallothionein contents, and serum levels of alanine aminotransferase and urea nitrogen. These effects of AuTM were compared to those of cadmium, since the latter metal has exhibited tissue and species differences in the induction of metallothionein. Benzo(a)pyrene hydroxylase and benzphetamine N-demethylase activities were not altered by AuTM in livers of rats and the three strains of mice. Benzo(a)pyrene hydroxylase activity was significantly decreased in rat kidney, whereas this enzyme activity was not affected in the kidneys of mice. In rats, AuTM caused a sevenfold induction in liver metallothionein, while in mice, liver metallothionein was induced twofold in Swiss-Webster mice and about fivefold in the inbred strains. AuTM caused minimal changes in renal metallothionein contents in the three strains of mice studied. Serum alanine amino-transferase, an indicator of hepatotoxicity, was not altered by AuTM in rats and mice studied. Blood urea nitrogen, an indicator of kidney dysfunction, was increased threefold in rats, but not in AuTM-treated mice. These data demonstrate that AuTM, a nephrotoxic agent in rats and humans, showed no nephrotoxic effects in the mouse strains studied here.
Rats were treated with nitrogen-containing phenanthrene (3,4-, 5,6-, or 7,8-benzoquinoline) or anthracene (acridine or quinacrine) derivatives at a dose of 75 mg/kg, daily for 3 days. The hepatic drug metabolizing enzyme response ranged from no induction (quinacrine) through low (5,6-benzoquinoline), intermediate (acridine), and high (3,4-benzoquinoline) magnitude increases of only phase II enzymes, to induction of both phase I and phase II enzymes (7,8-benzoquinoline). The phase I enzyme response of 7,8-benzoquinoline was an induction of CYP1A. All three benzoquinolines, but neither anthracene derivative, elevated NAD(P)H quinone oxidoreductase activity. A similar pattern but of lesser magnitude was seen with glutathione S-transferase activity. 3,4-Benzoquinoline was the only agent to significantly increase microsomal epoxide hydrolase activity (2,3-fold). Both 3,4- and 7,8-benzoquinoline increased UDP-glucuronosyltransferase activity toward 4-nitrophenol (40% and 70%, respectively), but only the 3,4-isomer increased activity toward morphine (75%), diclofenac (75%), and testosterone (23%), and only the 7,8-isomer increased activity toward chloramphenicol (105%). 3,4-Benzoquinoline elevated the hepatic mRNA concentration of UGT2B1 but not UGT1*6. Acridine treatment increased UDP-glucuronosyltransferase activity toward morphine (47%), 1-naphthol (28%), testosterone (19%), and estrone (19%). Quinacrine failed to elevate any UDP-glucuronosyltransferase activity and depressed activities toward testosterone and estrone by 20%. This study shows that some tricyclic aromatic compounds containing a single heterocyclic nitrogen atom have the potential for use as chemoprotective agents based upon their ability to selectively induce only phase II enzymes.
Journal of Biochemical ToxicologyVolume 11, Issue 2 p. 57-58 Enhancement in carcinogen-DNA binding in dietary iron deficiency and its reversal on iron supplementation N. Jayanthi Rao, N. Jayanthi Rao Food & Drug Toxicology Research Centre, National Institute of Nutrition, Jamaiosmania P.O. Hyderabad—500 007, IndiaSearch for more papers by this authorV. Jagadeesan, Corresponding Author V. Jagadeesan Food & Drug Toxicology Research Centre, National Institute of Nutrition, Jamaiosmania P.O. Hyderabad—500 007, IndiaFood & Drug Toxicology Research Centre, National Institute of Nutrition, Jamaiosmania P.O. Hyderabad—500 007, IndiaSearch for more papers by this author N. Jayanthi Rao, N. Jayanthi Rao Food & Drug Toxicology Research Centre, National Institute of Nutrition, Jamaiosmania P.O. Hyderabad—500 007, IndiaSearch for more papers by this authorV. Jagadeesan, Corresponding Author V. Jagadeesan Food & Drug Toxicology Research Centre, National Institute of Nutrition, Jamaiosmania P.O. Hyderabad—500 007, IndiaFood & Drug Toxicology Research Centre, National Institute of Nutrition, Jamaiosmania P.O. Hyderabad—500 007, IndiaSearch for more papers by this author First published: 1996 https://doi.org/10.1002/(SICI)1522-7146(1996)11:2<57::AID-JBT1>3.0.CO;2-TAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume11, Issue21996Pages 57-58 RelatedInformation
In the present investigation, we have studied the effects of aluminum (10 mg/kg of body weight per day, i.p.) and desferrioxamine (6 mg/kg of body weight per day, i.p.) alone and in combination for 4 weeks on the regulation of phosphorylation of neuronal proteins. A marked decrease in the biological activity of calmodulin was observed after aluminum treatment; however, in combination with desferrioxamine, a reversal in the levels of calmodulin, in terms of nmol cAMP hydrolyzed/min/mg protein, was observed. Exogenous addition of calmodulin had an inhibitory effect on calmodulin-mediated synaptosomal protein phosphorylation in aluminum-exposed animals. An almost complete reversal of this inhibition was observed following coexposure to aluminum and desferrioxamine. Cyclic AMP-dependent synaptosomal protein phosphorylation was, however, stimulated following aluminum exposure. Coadministration of desferrioxamine along with aluminum was found to mitigate the neurotoxic effect of aluminum.
Fumonisin B1 (FB1) causes equine leukoencephalomalacia, porcine pulmonary edema, and liver tumors and chronic nephritis in rats. To investigate mechanisms by which FB1 induces toxicity, effects of FB1 on cellular glutathione (GSH) redox status and GSH depletion on FB1 toxicity in pig kidney (LLC-PK1) cells were studied. Treatment of LLC-PK1 cells with 50 microM FB1 for 24 hours significantly decreased cellular GSH contents from 56 +/- 3.2 to 42.7 +/- 4.4 nmol/mg protein (p < 0.05) and increased the activities of glutathione reductase (GR) from 25.7 +/- 2.4 to 35.7 +/- 5.0 mumol NADPH/mg protein (p < 0.05). The activities of glutathione peroxidase (GSHpx), catalase, and Cu,Zn-superoxide dismutase (SOD) were not changed by this treatment. Treatment of LLC-PK1 cells for 12 hours with 0.1 mM buthionine sulfoximine (BSO), a selective inhibitor of the enzyme gamma-glutamylcysteine synthetase that catalyzes the rate-limiting reaction in de novo GSH synthesis, decreased cellular GSH levels to about 20% of that found in the control cells. The cells pretreated with 0.1 mM BSO for 12 hours were significantly sensitized to the FB1 cytotoxicity as determined by a long-term survival assay (p < 0.05). The results demonstrate that FB1 changes GSH redox cycle status in LLC-PK1 cells, and GSH may play a role in cytoprotection against FB1 toxicity.
The ability of trout to metabolize aniline in vitro in the presence of some divalent metal ions was investigated in the liver microsomes of rainbow trout, Salmo gairdneri. Trout liver microsomes were highly capable of catalyzing aniline hydroxylation to p-aminophenol with a specific activity of 0.068 nmoles/ min per mg of microsomal protein in potassium phosphate buffer, pH 7.4 at 25 degrees C. The activity of the aniline hydroxylase system was competitively inhibited by Hg+2, Ni+2, Cd+2, and Zn+2, while Cu+2 and Fe+3 seemed to inhibit the activity noncompetitively at 1 mM aniline concentrations. IC50 values at fixed aniline concentration were estimated to be 0.45 mM for Hg+2, Ni+2, and Cd+2, 1.8 mM for Zn+2 and Fe+3, and 1.3 mM for Cu+2. Eadie-Hofstee plots gave identical Vmax values of approximately 0.046 nmol/min per mg of protein while K(m) values were increased in the presence of Hg+2, Ni+2, Cd+2, and Zn+2, indicating competitive inhibition. Both K(m) and Vmax values were affected by Fe+3 and Cu+2, suggesting noncompetitive inhibition. Ki values extracted from the Dixon plots were determined to be 0.23, 0.43, and 0.65 mM for Hg+2, Ni+2, and Cd+2, respectively, providing the most effective inhibition on the aniline hydroxylase system among studied metal ions. The Ki values were much higher in the presence of others. The results indicate a selective inhibition of the aniline hydroxylase system of trout liver microsomes by divalent metal ions.
Diallyl sulfone (DASO2) is a metabolite of diallyl sulfide, a compound derived from garlic. The present study investigated the effect of DASO2 as a protective agent against acetaminophen (APAP)-induced hepatotoxicity in mice. Oral administration of DASO2 protected mice against the APAP-induced hepatotoxicity in a dose- and time-dependent manner. When administered 1 hour prior to, immediately after, or 20 minutes after a toxic dose of APAP, DASO2 at a dose of 25 mg/kg completely protected mice from development of hepatotoxicity, as indicated by liver histopathology and serum lactate dehydrogenase levels. Protective effect was observed when DASO2 at a dose as low as 5 mg/kg was given to mice 1 hour prior to APAP administration. Oral administration of DASO2 to mice 1 hour prior to a toxic dose of APAP significantly inhibited the APAP-induced glutathione depletion in the liver. DASO2 treatment also decreased the levels of oxidative APAP metabolites in the plasma without affecting the concentrations of nonoxidative APAP metabolites. In liver microsomes, 0.1 mM of DASO2 caused a 60% decrease in the rate of APAP oxidation to N-acetyl-p-benzoquinone imine, which was determined as glutathione conjugate. This inhibitory effect is mainly due to its inhibition of cytochrome P450 2E1 activity; with an IC50 value equal to 0.11 mM. DASO2 also slightly inhibited the activities of P450s 3A and 1A, with IC50 values > 5 mM. Furthermore, a single oral dose of DASO2 inactivated P450 2E1- and P450 1A-dependent activities in liver microsomes. The results suggest that the protective effect of DASO2 against APAP-induced hepatotoxicity is due to its ability to block acetaminophen bioactivation mainly by the inactivation and inhibition of P450 2E1.
The conditions required for growth and survival of cerebellar granule neurons in vitro are known to alter the developmental regulation of NMDA receptor subunit mRNA. In the present report, we have examined the functional and pharmacological characteristics of NMDA receptors on cerebellar granule neurons at 12 days in culture (12 DIC). Under open-channel conditions in extensively washed membranes, [3H]MK-801 labeled a uniform population of sites (Kd = 3.2 +/- 0.3 nM) in a saturable manner (Bmax = 416 +/- 18 fmol/mg); however, biexponential association and dissociation kinetics indicated the possible existence of at least two NMDA receptor populations that differ in pharmacological properties. The kinetically derived equilibrium dissociation constants for the high- and low-affinity binding components were 0.56 and 771 nM, respectively. The equilibrium competition analysis of MK-801 and other channel-blocking compounds as displacers of [3H]MK-801 revealed the presence of high- and low-affinity binding sites with relative apportionments of 70% and 30%, respectively. The rank-order potency profile of competitor binding at the high-affinity site was (+)-MK-801 > TCP > dextrorphan > dextromethorphan > (+)-ketamine. When tested for the ability to protect 12 DIC cerebellar granule neurons from acute glutamate-induced toxicity, the neuroprotective rank-order potency of these compounds was MK-801 > TCP > dextrorphan > (+)-ketamine > dextromethorphan, which correlated significantly with the high-affinity competition binding profile and thus established the role of NMDA receptors in glutamate toxicity. The findings of these experiments indicate that NMDA receptors on 12 DIC cerebellar granule neurons are a heterogenous population that functionally mediate glutamate-induced neurotoxicity. The heterogenous [3H]MK-801 binding sites may represent NMDA receptor channels composed of different subunits.
Tricyclic antidepressants (TCAs) have been shown to induce apoptosis in human lymphocytes. In the present report, we investigated in parallel the regulation of the three oncogenes bcl-2, c-myc, and Fas. A reduction in c-myc and bcl-2 levels of 35-40% and 22-27%, respectively, was observed. On the other hand, Fas expression on the outer surface of the plasma membrane was increased up to 31%. In conclusion, bcl-2, c-myc, and Fas are undergoing dysregulation due to TCA-induced apoptosis.
The adducts produced in vitro by the reactive metabolites of [14C]-chloroform with total phospholipids (PLs) of freshly isolated hepatocytes have been characterized. The radical metabolite formed several adducts with all the major PL classes. These adducts seemed very likely to result from the unspecific attack of the radical on the PL fatty acyl chains. [14C]-Chloroform-derived phosgene caused the formation of a single PL adduct characterized by a ratio 14C:P of 1:4. This adduct was tentatively identified as an adduct of phosgene with two molecules of cardiolipin.
Cyanide-induced neurotoxicity is associated with altered cellular Ca2+ homeostasis resulting in sustained elevation of cytosolic Ca2+. In order to characterize the effect of cyanide on intracellular signaling mechanisms, the interaction of KCN with the inositol 1,4,5-trisphosphate Ca2+ signaling system was determined in the PC12 cell line. KCN in the concentration range of 1.0-100 microM produced a rapid rise in intracellular IP3 levels (peak level occurred within 60 sec); 10 microM KCN elevated intracellular levels of IP3 to 148% of control levels. This response was mediated by phospholipase C (PLC) since U73122, a specific PLC inhibitor, blocked the response. Removal of Ca2+ from the incubation medium and chelation of intracellular Ca2+ with BAPTA partially attenuate the cyanide-stimulated IP3 generation, showing that the response is partially Ca2+ dependent. Also, treatment of cells with nifedipine or LaCl3, Ca2+ channel blockers, partially blocked the generation of IP3. This study shows that cyanide in concentrations as low as 1 microM stimulates IP3 generation that may be mediated by receptor and nonreceptor IP3 production since they have differential dependence on Ca2+. It is proposed that this response is an early intracellular signaling action that can contribute to altered Ca2+ homeostasis characteristic of cyanide neurotoxicity.
The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on lipid peroxidation, 3H-Me-glucose (3H-Me-glu), and 14C-dehydroascorbic acid (14C-DHA) uptakes were studied in adipose tissue of male guinea pig. Under in vitro test conditions, using isolated adipose tissue in a culture medium (explant culture), TCDD reduced the uptake of 3H-Me-glu and 14C-DHA in a dose- and time-dependent fashion. The IC50 values of TCDD's action were 0.04 and 2 nM on 14C-DHA and 3H-Me-glu uptakes, respectively. TCDD (10 nM) also suppressed glucose transporting activity within 15 minutes in explant-cultured adipocytes. Cytochalasin B (CB) and nonlabeled D-glucose inhibited 14C-DHA uptake also in a dose-dependent manner. In addition, TCDD was found to induce lipid peroxidation in explant-cultured adipose tissue. This effect of TCDD was similar to that of a typical lipid peroxidation inducer, CCl4, and it was dose and time dependent. TCDD caused a statistically significant rise in lipid peroxidation at a concentration as low as 0.1 nM after 60 minutes of treatment in explant culture. Unexpectedly, the Ah receptor partial antagonists, 4,7-phenanthroline and alpha-naphthoflavone, did not fully antagonize TCDD-induced lipid peroxidation in explant-cultured adipocytes. In vivo treatment of TCDD also induced lipid peroxidation. Among seven organs of male guinea pig tested, the levels of lipid peroxidation in adipose tissue and in liver increased at 1 and 40 days following a single i.p. dose of TCDD (1 microgram/kg). The results of an in vivo time-course study indicated that such an effect of TCDD was most pronounced after 40 days of treatment. Finally, we have tested the protective role of some antioxidants on TCDD-induced lipid peroxidation under explant-culture conditions. The results indicated that DHA, but not ascorbic acid, could completely abolish TCDD-induced lipid peroxidation. The protective effect of DHA on TCDD-induced lipid peroxidation was stronger than that of alpha-tocopherol and uric acid, and this effect was blocked by CB. We conclude from these studies that TCDD acts in this guinea pig tissue through two different routes: one is the Ah receptor-dependent route causing the reduction of the level of glucose transporters and subsequent decrease of cellular uptake of DHA and the other, the Ah receptor-independent route causing the overall lipid peroxidation. Nevertheless, it appears likely that both events are antagonized by DHA.
Respiratory activity in hepatic mitochondria have been examined following administration of the carcinogen aflatoxin, (AFB1) to rats. Measurement in isolated mitochondria of respiration rates in presence of ADP (state 3) and after its depletion (state 4) revealed that these rates were not significantly altered in livers of rats obtained 4-8 hours after single injection of AFB1 (7 mg/kg of body weight). After 12-24 hours, however, a generalized inhibition in state 3 respiration rate and ADP phosphorylation rate had been evident with several FAD- and NAD-linked oxidizing substrates. But the ADP:0 ratio did not show any alteration. State 4 respiration rates, on the other hand, were increased remarkably (38-94% depending on substrate used), thereby recording in each case a decrease in respiratory control ratio (state 3:state 4 ratio), indicating probable damage to mitochondrial membrane as a result of AFB1 ingestion. This was also evident from greater basal ATPase and Mg(2+)-ATPase activities and low total ATPase activity. After 48-72 hours of AFB1 treatment, the respiratory rates as well as the ATPase activities returned to normal levels, suggesting probable recovery of mitochondrial functions from the toxic effects of AFB1.
Exposure of HeLa cells to different concentrations of the antineoplastic drug paclitaxel resulted in a loss of cell viability that was dependent on the concentration and time of exposure to the drug. This phenomenon was associated with the appearance of nuclear morphology typical of apoptosis and DNA breakage into a "ladder" pattern of discrete fragments of nucleosomal size. The induction of cell death was dependent on the serum concentration of the culture media, repressed by pretreatment with a cAMP-dependent protein kinase (PKA) inhibitor, and enhanced by increasing the cell proliferation with previous exposure to a cAMP-analog and a protein kinase-C (PKC) inducer. The proliferative index modifies the effect of taxol on HeLa cells, probably by means of a more rapid accumulation of cells in the G2/M cycle blockage point, although a direct participation of PKA and PKC should not be excluded.
We have examined the effects of mercuric chloride on renal glomerular structure. Isolated glomeruli from mercury-treated rats (HgCl2, 5 mg/kg body wt, s.c.) 1 hour post injection presented a diminished cross-sectional area as compared with control glomeruli [control (micron2) = 26,310 +/- 2,545, HgCl2 (micron2) = 18,474 +/- 1,828] and higher glomerular calcium content (control = 23 +/- 6 nmoles/mg prot, HgCl2 = 43 +/- 7 nmoles/mg prot). Renal sections prepared for immunohistochemical and histochemical analysis showed larger deposits of fibronectin and lipids and enhanced cellularity in glomerular structures from HgCl2-treated rats. Moreover, mieloperoxidase activity measured in isolated glomeruli were also increased as compared with control preparations [MPO (U/mg prot): control = 59 +/- 7, HgCl2 = 134 +/- 10]. When the animals were studied 24 hours post HgCl2 injection, glomerular cross-sectional area values were not different from control values (25,276 +/- 1,983 micron2), while calcium contents were higher than values observed 1 hour after treatment (92 +/- 9 nmoles/mg prot). A similar pattern was observed in fibronectin deposits. Hypercellularity in glomerular structures and the higher mieloperoxidase levels were maintained at this time (MPO HgCl2-rats 24 h = 148 +/- 31 U/mg prot). The effects observed in this study are consistent with an inflammatory response in the glomerular structure of HgCl2-treated rats that could explain the altered renal function described in previous reports in our laboratory.
The cytochrome P-450 (CYP) mediated hydroxylation of testosterone to 6 beta-, 7 alpha-, and 16 alpha-hydroxytestosterone (b beta-, 7 alpha-, and 16 alpha-OHT) and the dealkylation of ethoxycoumarin to 7-hydroxycoumarin (ECOD) and ethoxyresorufin to resorufin (EROD) were used to probe changes in CYP monooxygenase activities in liver microsomes from rats treated with the androgen receptor antagonist, zanoterone (Z). Phenobarbital (PB) and beta-naphthoflavone (beta-NF) were used as comparators. There were sex-related differences in the constitutive CYP activities and in the responses of CYP activities to Z. The greatest effect of Z administration was on 6 beta-OHT activity: It was increased up to 5.2-fold in males and 13.9-fold in females (Z high dose). The effect was larger than the produced by PB or beta-NF (< or = threefold increases). Z (high dose), PB, and beta-NF increased ECOD to a similar extent, e.g., about 1.3-fold in males and 1.2-2.9-fold in females. beta-NF increased EROD (11.2-fold males, 6.2-fold females) more than PB (3.4- to 4.6-fold) or Z (1.3- to 1.7-fold). Since hydroxylation of testosterone at the 6 beta position in rats and humans is catalyzed primarily by CYP isoforms from the 3A subfamily, the increase in 6 beta-OHT suggests that Z induced CYP 3A activity. These findings were confirmed with Western immunoblots with probes for rat CYP 1A1, 2B1/2, 2E1, 3A, and 4A. Z produced a three-to fourfold increase in the 3A isoform for both male and female rats. Results from this study suggest that in a clinical setting, Z therapy has the potential to induce CYPs of the 3A subfamily and in so doing alter the metabolism and clearance of drugs that are substrates for the 3A subfamily.
3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three alpha-keto acids (alpha-ketobutyrate, alpha-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the alpha-keto acid. However, it has also been proposed that alpha-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three alpha-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three alpha-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant Ki was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I50, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three alpha-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanism of the alpha-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate.
