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Biomedical subjects

G J Mulder

Publications and source records attributed to G J Mulder.

At least 19 recordsLinked to original sources
PubMed1997

Effect of coexposure to methyl ethyl ketone (MEK) on n-hexane toxicokinetics in human volunteers.

In order to study the effects of methyl ethyl ketone (MEK) on the toxicokinetics of n-hexane and, in particular, the formation of 2,5-hexanedione from n-hexane in humans, volunteers were exposed to n-hexane (approx. 60 ppm, 2.4 microM in the inhaled air) with or without simultaneous inhalatory coexposure to MEK for 15.5 min. The concentration-time course of n-hexane (in exhaled alveolar air) and its neurotoxic metabolite, 2,5-hexanedione (in serum), were studied. The concentration-time courses obtained after exposure to n-hexane alone were compared with those obtained after coexposure to 200 or 300 ppm MEK in the same volunteer on the same day. No effect of MEK was observed on the concentration-time course of exhaled n-hexane. The concentration-time course of the metabolite, 2,5-hexanedione, revealed a decrease in the rate of formation of 2,5-hexanedione (about three-fold) after coexposure to MEK. Furthermore, the time to reach the peak concentration was increased from 18 to 30 min after the start of exposure. These changes in the concentration-time course of 2,5-hexanedione caused by MEK are most likely the result of inhibition of the biotransformation of one of the intermediate steps in the conversion of n-hexane to 2,5-hexanedione. These results indicate that the interaction of n-hexane and MEK leads to a decreased concentration of the neurotoxic metabolite 2,5-hexanedione (after short-term, acute exposure).

Administration, Inhalation↗
PubMed1997

p53 protein expression by hepatocarcinogens in the rat liver and its potential role in mitoinhibition of normal hepatocytes as a mechanism of hepatic tumour promotion.

The tumour suppressor gene p53 is expressed in response to DNA-damage; its protein product blocks cells in the G1-phase of the cell cycle. This gives cells additional time to repair their DNA-damage. However, it may trigger apoptosis if damage is too high. Loss of p53 function appears to be an important step in carcinogenesis because 50% of human tumours have lost functional p53. In order to study the role of p53 in experimental hepatocarcinogenesis, we determined the expression of p53 in rat liver in response to various hepatocarcinogenic and hepatotoxic compounds. Administration of hepatocarcinogenic compounds increased p53 protein levels in the liver as detected by immunoprecipitation followed by SDS-PAGE and Western blotting with ECL-detection. The hepatocarcinogens included N-hydroxy-2-acetylaminofluorene, aflatoxin B1, and diethylnitrosamine. Their structural analogues N-hydroxy-4-acetylaminobiphenyl and ethyl methane-sulphonate which are not hepatocarcinogenic, did not induce p53. Also, two hepatotoxic compounds (carbon tetrachloride, D-galactosamine) did not induce p53. Other compounds that induced p53 in the rat liver were 2-aminofluorene (administered by drinking water for two weeks) and tris-(2,3-dibromopropyl)phosphate. Benzo[a]pyrene did not induce p53. N-Hydroxy-2-acetylaminofluorene, aflatoxin B1, and diethylnitrosamine are potent hepatic tumour promoters. At the same time, they induce p53 protein expression and inhibit proliferation of normal hepatocytes. Because this is not observed with non-hepatocarcinogenic analogues, it suggests an involvement of p53 expression in hepatic tumour promotion. A possible mechanism is discussed.

Alanine Transaminase↗
PubMed1997

Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain.

Cisplatin-induced nephrotoxicity was studied in porcine proximal tubular cells, focusing on the relationship between mitochondrial damage, reactive oxygen species (ROS) and cell death. Cisplatin specifically affected mitochondrial functions: complexes I to IV of the respiratory chain were inhibited 15 to 55% after 20 min of incubation with 50 to 500 microM, respectively. As a result, intracellular ATP was decreased to 70%. The mitochondrial glutathione (reduced form) (GSH)-regenerating enzyme GSH-reductase (GSH-Rd) activity was reduced by 20%, which contributed to a 70% reduction of GSH levels and ROS formation. The residual electron flow through the mitochondrial respiratory chain was the source of ROS because additional inhibition of the complexes I to IV reduced ROS formation. Because cisplatin affects both GSH-Rd and complexes I to IV, cells were incubated with N,N'-bis(2-chloroethyl)-N-nitrosourea (inhibitor of GSH-Rd) and inhibitors of the different complexes. Only N,N'-bis(2-chloroethyl)-N-nitrosourea with rotenone (complex I inhibitor) induced ROS formation, which indicates that inhibition of complex I and inhibition of the GSH-Rd is probably the cause of ROS formation. However, the resulting ROS is not the cause of cell death because diphenyl-p-phenylene-diamine and deferoxamine, which completely prevented ROS, could not prevent cell death. Similarly, the antioxidants did not completely prevent the decrease in activity of complexes I to IV, ATP or GSH levels. In conclusion, ROS formation does occur during cisplatin-induced toxicity, but it is not the direct cause of cell death.

Adenosine Triphosphate↗
PubMed1996

Lack of glutathione conjugation of melphalan in the isolated in situ liver perfusion in humans.

Tumor cell resistance against melphalan (LPAM) has been associated with increased cellular reduced glutathione (GSH) levels and glutathione S-transferase activity. Therefore, GSH conjugation of LPAM has been hypothesized to be a key factor in tumor cell resistance. In the present study, we evaluated GSH conjugation of LPAM by the perfused liver in patients with colorectal cancer metastases undergoing a Phase II study of isolated liver perfusion as well as in the rat. To evaluate whether LPAM-GSH conjugates were synthesized in the rat in vivo, LPAM was infused i.v. at a rate of 2.0 micromol/kg/min. In bile samples obtained during the infusion, two major GSH conjugates were identified by mass spectrometry: mono-hydroxy-mono-GSH-LPAM and di-GSH-LPAM. The maximum biliary excretion rate of these two conjugates accounted for only 1.3% of the LPAM infusion rate. In bile or perfusate samples from patients treated for 60 min initially with 0.3 mM LPAM in the perfusion medium via isolated liver perfusion (200 mg LPAM in approximately 2 liters perfusion medium), none of the above-mentioned conjugates were detected. When comparable rat liver perfusions were performed initially with 66 microM or 0.66 mM LPAM in the perfusion medium, bile samples did contain GSH-LPAM conjugates; the cumulative biliary excretion of the two conjugates amounted to 0.4 and 0.2% of the LPAM dose, respectively. These data suggest that both in rats and humans, hepatic GSH conjugation plays a very minor (if any) role in the elimination of LPAM and, therefore, that modulation of GSH levels is unlikely to affect the rate of elimination of this drug.

Animals↗
PubMed1996

Isoenzyme-selective inhibition of glutathione conjugation in vivo: selective inhibition of the conjugation of S-2-Bromoisovalerylurea in the rat.

Glutathione S-transferases (GSTs) play a major role in the (de-)toxification of many endogenous and xenobiotic substrates. To assess their contribution in (de-)toxification, specific in vivo inhibitors that ideally are selective for a single isoenzyme of GST are required. In the present study, selective inhibition of the alpha class GST by the glutathione analog (R)-5-ethyloxycarbonyl-2-gamma-(S)-glutamylamino-N-2-hept ylpentamide (Et-R-Hep) was studied. In rat liver cytosol and in isolated rat hepatocytes, only the conjugation of the (S)-enantiomer of (RS)-2-bromoisovalerylurea (BIU), which is conjugated mainly by alpha class GST 2-2 (Te Koppele et al., Biochem. J. 252:137-142, 1988), was inhibited by Et-R-hep. The conjugation of (R)-BIU, which is mainly catalyzed by mu class GSTs 3-3 and 4-4, was unaffected. In anesthetized rats to which an infusion of (RS)-BIU was administered, the biliary excretion of the glutathione conjugate of (S)-BIU was inhibited by up to 67% after administration of Et-R-hep (an i.v. bolus dose of 200 mu mol/kg followed by an infusion of 6.7 mu mol/min/kg for 30 min). The extent of inhibition decreased gradually to reach 40% at the end of the experiment (4 hr after administration of the inhibitor). The conjugation of (R)-BIU was unaffected. Thus, the inhibitor Et-R-Hep shows preferential inhibition of the alpha-GST substrate (S)-BIU. Although Et-R-Hep is not specific for alpha class GST, it may be used to assess the role of this class of GST in (de)-toxification and conjugation in vivo.

Animals↗
PubMed1995

Glutathione analogues as novel inhibitors of rat and human glutathione S-transferase isoenzymes, as well as of glutathione conjugation in isolated rat hepatocytes and in the rat in vivo.

Inhibitors of rat and human Alpha- and Mu-class glutathione S-transferases that effectively inhibit the glutathione (GSH) conjugation of bromosulphophthalein in the rat liver cytosolic fraction, isolated rat hepatocytes and in the rat liver in vivo have been developed. The GSH analogue (R)-5-carboxy-2-gamma-(S)-glutamylamino-N-hexylpentamide [Adang, Brussee, van der Gen and Mulder (1991) J. Biol. Chem. 266, 830-836] was used as the lead compound. To obtain more potent inhibitors, it was modified by replacement of the N-hexyl moiety by N-2-heptyl and by esterification of the 5-carboxy group with ethyl and dodecyl groups. In isolated hepatocytes, the branched N-2-heptyl derivatives were stronger inhibitors of GSH conjugation of bromosulphophthalein than the N-hexyl derivatives. The ethyl ester compounds were more efficient than the corresponding unesterified derivatives. The dodecyl ester of the N-2-heptyl analogue was the most effective inhibitor in isolated hepatocytes, but was relatively toxic in vivo. However, the corresponding ethyl ester was a potent in vivo inhibitor: GSH conjugation of bromosulphophthalein (as assessed by biliary excretion of the conjugate) was decreased by 70% after administration of a dose of 200 mumol/kg. The isoenzyme specificity of the inhibitors towards purified rat and human glutathione S-transferases was also examined. The unesterified compounds were more potent than the esterified analogues, and inhibited Alpha- and Mu-class isoenzymes of both rat and human glutathione S-transferase (Ki range 1-40 microM). Other GSH-dependent enzymes, i.e. GSH peroxidase, GSH reductase and gamma-glutamyltranspeptide, were not inhibited. Thus (R)-5-ethyloxycarbonyl-2-gamma-(S)-glutamylamino-N-2-hept ylpentamide, the in vivo inhibitor of GSH conjugation, may be useful in helping to assess the role of the Alpha and Mu classes of glutathione S-transferases in cellular biochemistry, physiology and pathology.

Animals↗
PubMed1995

Deuterium isotope effect on the metabolism of the flame retardant tris(2,3-dibromopropyl) phosphate in the isolated perfused rat liver.

The metabolism of tris(2,3-dibromopropyl) phosphate (Tris-BP) was compared with that of completely deuterated Tris-BP (D15-Tris-BP) in an isolated, recirculating rat liver perfusion system in order to determine the relative quantitative importance of two different biotransformation pathways of Tris-BP: (i) cytochrome P450-mediated metabolism and (ii) GSH S-transferase-mediated metabolism. To accomplish this we quantitated the biliary excretion of S-(3-hydroxypropyl)glutathione (GSOH) as a marker metabolite for cytochrome P450-mediated metabolism and that of S-(2,3-dihydroxypropyl) glutathione (GSOHOH) as a marker metabolite for GSH S-transferase-mediated metabolism. Complete deuterium substitution of Tris-BP significantly decreased the formation of GSOH, whereas there was no effect on the formation of GSOHOH. Because our previous studies showed a large decrease in genotoxicity of D15-Tris-BP compared to Tris-BP, the present results support our hypothesis that cytochrome P450-mediated metabolism is responsible for the genotoxic effects of Tris-BP in the rat liver.

Animals↗
PubMed1995

Glutathione conjugation of bromosulfophthalein in relation to hepatic glutathione content in the rat in vivo and in the perfused rat liver.

The relation between the rate of glutathione (GSH) conjugation and hepatic GSH content was studied in the rat in vivo and the in situ single-pass-perfused rat liver preparation with bromosulfophthalein (BSP) as the model substrate. The biliary excretion of the BSP-GSH conjugate and the hepatic GSH content were monitored simultaneously during intravenous infusions with BSP in the rat in vivo, and during liver perfusions with BSP-containing perfusion medium. Rats were pretreated with single or multiple doses of buthionine sulfoximine, an inhibitor of the de novo synthesis of GSH. Surprisingly, the excretion of the BSP-GSH conjugate was sustained at a high rate, despite a virtually complete depletion of hepatic GSH, both in the rat in vivo as well as in the perfused rat liver. The results indicate that GSH was still available for conjugation with BSP after apparent depletion of the hepatic GSH pool, presumably because of a residual de novo synthesis of GSH in the liver. Despite the multiple pretreatment with buthionine sulfoximine, the de novo GSH synthesis was sufficient to sustain a high rate of GSH conjugation of BSP. The cosubstrate-Km for GSH conjugation of BSP in the liver was estimated to be very small (approximately 0.3 mumol/g): the excretion rate of the BSP-GSH conjugate was only impaired at minimal hepatic GSH levels.

Animals↗
PubMed1995

Disposition of the bromosulfophthalein-glutathione conjugate in the isolated perfused rat kidney.

Renal elimination of the bromosulfophthalein-glutathione conjugate (BSP-GSH) after its i.v. administration in the rat in vivo is negligible. In our study we wanted to establish whether the high albumin-binding of BSP-GSH constitutes the major restrictive factor toward the urinary excretion of the compound. The renal disposition of BSP-GSH was studied in the isolated rat kidney during perfusions with or without albumin in the perfusate. The urinary clearance of BSP-GSH in the absence of albumin was very low (< 60 microliters/min) as compared to the inulin clearance (approximately 300 microliters/min). This indicates that albumin-binding is not the major reason for the low urinary clearance of BSP-GSH. Addition of albumin to the perfusate further decreased the urinary excretion by 60%. BSP-GSH is metabolized by the kidney into two major metabolites: the cysteinylglycine conjugate and the di-glutathione conjugate. Both metabolites appear in perfusate, which suggests that BSP-GSH undergoes tubular (re-)uptake. The di-glutathione conjugate is further metabolized to the di-cysteinylglycine conjugate. The di-glutathione conjugate and the di-cysteinylglycine conjugate are the major urinary components and the urinary elimination of BSP-GSH may depend on their formation. Inhibition of gamma-glutamyl transpeptidase activity with acivicin largely prevented the degradation to the cysteinylglycine and dicysteinylglycine conjugates of BSP. The total rate of urinary excretion, however, was only slightly lowered by acivicin. Apparently, cleavage of the gamma-glutamyl moiety is not relevant for the total urinary elimination of BSP-GSH.

Albumins↗
PubMed1995

Metabolic activation and DNA adduct detection of PhIP in dogs, rats, and humans in relation to urinary bladder and colon carcinogenesis.

The metabolic activation of the heterocyclic amine carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was examined in dogs and rats as models for urinary bladder and colon carcinogenesis, respectively. The results indicate that unconjugated N-OH-PhIP is not excreted in the urine after oral dosing with PhIP and that the two isomeric N-glucuronides of N-OH-PhIP, which are formed as major metabolites, are stable under acidic conditions. These data suggest that PhIP is unlikely to serve as a urinary bladder carcinogen in either species. Using metabolic inhibitors, bile duct ligation, and intravenous dosing studies, a new hypothesis for colorectal carcinogenesis is proposed involving N-oxidation of PhIP by hepatic cytochrome P-4501A2 (CYP1A2) and O-acetylation by the polymorphic acetyltransferase (NAT2). The resulting N-hydroxy and N-acetoxy metabolites both appear to be transported through the circulation to the colon mucosa, forming covalent DNA adducts. Glucuronidation and reaction with glutathione appear to serve as detoxification pathways. In humans, individuals who are phenotypically rapid metabolizers for both CYP1A2 and NAT2 are significantly higher (p = 0.0015) in colorectal cancer/poly cases vs. controls; and PhIP-DNA adducts can be detected in human colon samples. These studies provide strong evidence that PhIP and other heterocyclic amines play an important role in the etiology of human colorectal cancer.

Animals↗
PubMed1994

Role of mitochondrial Ca2+ in the oxidative stress-induced dissipation of the mitochondrial membrane potential. Studies in isolated proximal tubular cells using the nephrotoxin 1,2-dichlorovinyl-L-cysteine.

The relationship between mitochondrial Ca2+, oxidative stress, and a dissipation of the mitochondrial membrane potential (delta psi) was investigated in proximal tubular kidney cells. Freshly isolated proximal tubular cells from rat kidney were exposed to the nephrotoxin 1,2-dichlorovinyl-L-cysteine (DCVC). DCVC stimulated the formation of hydroperoxides as determined by flow cytometry using the hydroperoxide-sensitive compound dichlorofluorescein. This was prevented by the antioxidant diphenylphenylenediamine (DPPD) and the iron chelator desferrioxamine. Studies in individual cells with video-intensified fluorescence microscopy showed that a DCVC-induced increase in the intracellular free calcium concentration ([Ca2+]i) was accompanied by an increase in the mitochondrial free calcium concentration ([Ca2+]m). The latter increase was selectively prevented by an inhibitor of the mitochondrial calcium uniporter, ruthenium red (RR). Chelation of cellular Ca2+ with EGTA acetoxymethyl ester (EGTA/AM) completely prevented the formation of hydroperoxides, whereas inhibition of the uptake of Ca2+ by the mitochondria with RR reduced it. This indicates that the increase in [Ca2+]m is important for the induction of oxidative stress by DCVC. DPPD and desferrioxamine did not protect against a DCVC-induced increase in [Ca2+]i and [Ca2+]m, indicating that oxidative stress is the consequence rather than the cause of the cellular calcium perturbations. DCVC decreased delta psi and caused cell death; both effects were clearly delayed by EGTA/AM and RR, although they could not prevent a decrease in delta psi. The latter decrease was completely prevented by inhibition of the beta-lyase-mediated metabolism of DCVC with aminooxyacetic acid. Like EGTA/AM, inhibition of oxidative stress with DPPD and desferrioxamine delayed the decrease in delta psi. This strongly suggests that the decrease in delta psi caused by metabolites of DCVC directly is potentiated by Ca(2+)-dependent DCVC-induced hydroperoxide formation. The importance of both hydroperoxide formation and mitochondrial damage in DCVC-induced cell killing is discussed.

Animals↗
PubMed1994

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes.

The food-borne carcinogenic and mutagenic heterocyclic aromatic amines undergo bioactivation to the corresponding N-hydroxy (OH)-arylamines and the subsequent N-glucuronidation of these metabolites is regarded as an important detoxification reaction. In this study, the rates of glucuronidation for the N-OH derivatives of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by liver microsomal glucuronosyltransferase were compared to that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl (N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-amino-biphenyl (N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronic acid (UDPGA)-dependent glucuroidation of N-OH-IQ, N-OH-PhIP, N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%, respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg). Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidation of N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20% and 10%, respectively of that measured for N-OH-DMABP (11.2 nmol/min/mg); activity towards N-OH-MeIQx was not detected. Two glucuronide(s) of N-OH-PhIP, designated I and II, were separated by HPLC. Conjugate II was found to be chromatographically and spectrally identical with a previously reported major biliary metabolite of PhIP in the rat, while conjugate I was identical with a major urinary metabolite of PhIP in the dog. Hepatic microsomes from rat, dog and human were found to catalyze the formation of both conjugates. The rat preferentially formed conjugate II (I to II ratio of 1:15), while the dog and human formed higher relative amounts of conjugate I (I to II ratio of 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardment mass spectrometry of conjugates I and II gave the corresponding molecular ions and showed nearly identical primary spectra. However, collision-induced spectra were distinct and were consistent with the identity of conjugates I and II as structural isomers. Moreover, the UV spectrum of conjugate I exhibited a lambda max at 317 nm and was essentially identical to that of N-OH-PhIP, while conjugate II was markedly different with a lambda max of 331 nm. Both conjugates were stable in 0.1 N HCl and were resistant to hydrolysis by rat, dog and human liver microsomal beta-glucuronidases.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues.

The food-borne mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces tumors in colon of male rats and has been implicated in the etiology of human cancers, particularly colorectal cancer. This study was conducted to examine: (1) the biliary and/or circulatory transport of N-hydroxy-PhIP and its N-glucuronides, N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximate and ultimate carcinogenic metabolites of PhIP; (3) the potential role of glutathione in modulating PhIP-DNA adduct formation. PhIP-DNA adducts, measured by the 32P-postlabeling method, were highest in the pancreas (361 adducts/10(8) nucleotides or 100%), followed by colon (56%), lung (28%), heart (27%) and liver (2%), at 24 h after a single oral dose of PhIP (220 mumol/kg) to male rats. In each tissue examined, we observed two major adducts, each of which accounted for 35-45% of the total, and one minor adduct, which represented about 10-20% of the total. One of the major adducts was identified as N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine by chromatographic comparisons with an authentic standard. The major urinary metabolites of PhIP in these rats were 4'-hydroxy-PhIP and its glucuronide and sulfate conjugates, followed by N-hydroxy-PhIP N3-glucuronide, N-hydroxy-PhIP N2-glucuronide and unchanged PhIP. In bile duct-ligated rats, the urinary excretion of the N-OH-PhIP N3-glucuronide was increased two-fold, but there was no effect on PhIP-DNA adduct formation in the colon, heart, lung, pancreas or liver. 2,6-Dichloro-4-nitrophenol, which strongly inhibits arylsulfo-transferase-mediated DNA binding in vivo, had no effect on PhIP-DNA adduct levels in liver or in extrahepatic tissues. Pretreatment of rats with buthionine sulfoximine, which results in hepatic glutathione depletion, caused a five-fold increase in adduct formation in the liver. Intravenous administration (10 mumol/kg) of N-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels of PhIP-DNA adducts in each of the extrahepatic tissues examined. Adduct levels ranged from two- to six-fold higher (for N-hydroxy-PhIP) and four- to 28-fold higher (for N-acetoxy-PhIP) as compared to that after an i.v. dose of the parent compound, indicating that these two bioactivated derivatives of PhIP are sufficiently stable to be transported through the circulation to extrahepatic tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes.

The potent rat colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), unlike other food-borne heterocyclic amines, does not induce tumors in rat liver. This correlates with an extremely low level of PhIP-DNA adducts formed in this tissue, and together these observations suggest that PhIP is efficiently detoxified in the liver. In order to identify possible detoxification mechanisms, we assessed the effect of inhibition of glucuronidation, glutathione (GSH) conjugation and sulfation on PhIP metabolism and PhIP-induced DNA damage in rat hepatocytes. Hepatocytes isolated from rats pretreated with Aroclor 1254 metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIP N3-glucuronide and N-hydroxy-PhIP N2-glucuronide were major and minor metabolites respectively. 32P-Postlabeling analysis of DNA from the PhIP-treated hepatocytes indicated the presence of two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP, and one minor adduct. There was no unscheduled DNA synthesis (UDS) in these cells. However, pretreatment of the hepatocytes with 1-bromoheptane and buthionine sulfoximine, which depletes GSH and prevents its resynthesis, resulted in a 15-fold increase in the formation of PhIP-DNA adducts, as well as in a high level of UDS. GSH depletion had no effect on the formation of detectable PhIP metabolites. Hepatocyte pretreatment with D-galactosamine, which inhibits glucuronidation, increased the formation of DNA adducts two-fold and UDS was increased similarly. D-Galactosamine decreased the formation of the two N-glucuronides of N-hydroxy-PhIP by 50-60%, but had no effect on other metabolites. Pentachlorophenol, which strongly inhibits sulfotransferases, decreased adduct formation slightly, but had essentially no effect on UDS or on the formation of PhIP metabolites. These results indicate that metabolic conjugation pathways involving GSH and glucuronidation may play an important role in protecting rat liver against PhIP carcinogenesis.

Animals↗
PubMed1994

In vivo and in vitro detachment of proximal tubular cells and F-actin damage: consequences for renal function.

We investigated the relationship between F-actin damage and cell detachment using nephrotoxic L-cysteine S-conjugates. In vivo S-(1,2-dichlorovinyl)-L-cysteine (DCVC) induced loss of F-actin in the S3 segment of the proximal tubule in the outer stripe of the outer medulla, which was associated with loss of the brush border and loss of cells from the basement membrane. In vitro DCVC caused the detachment of primary cultured rat renal proximal tubular cells (PTC), which was clearly associated with F-actin damage. Disorganization of F-actin correlated with an increase in cellular levels of G-actin, indicating depolymerization of F-actin. Cell detachment was preceded by a complete loss of the alpha-actinin binding protein talin from the focal adhesions, which was directly associated with F-actin disorganization. Inhibition of formation of highly reactive metabolites from L-cysteine S-conjugates by L-cysteine-S-conjugate beta-lyase completely prevented both F-actin damage and cell detachment by DCVC. Although inhibition of DCVC-induced lipid peroxidation and reduction of intracellular free calcium by N,N'-diphenyl-p-phenylenediamine and the acetoxymethyl ester of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, respectively, clearly prevented cell death, no protection was observed against the DCVC-induced F-actin disorganization, talin redistribution, and cell detachment. Also, F-actin damage was unrelated to changes in the energy status of the PTC, since cellular ATP content was unaffected. The data clearly demonstrate a close relationship between F-actin damage, disturbances of focal adhesions, and cell detachment. In addition, different molecular pathways are involved in the cell detachment caused by F-actin disorganization and initiation of cell death.

Actins↗
PubMed1993

Stereoselectivity of human liver and intestinal cytosolic fractions as well as purified human glutathione S-transferase isoenzymes towards 2-bromoisovalerylurea enantiomers.

Glutathione (GSH) conjugation of 2-bromoisovalerylurea (BIU) enantiomers is stereoselective in humans in vivo. Administration of racemic BIU results in a higher plasma elimination and urinary excretion of R-BIU and its mercapturate, respectively, than of S-BIU and its mercapturate. In order to relate the in vivo BIU pharmacokinetics to the activity of glutathione S-transferase (GST) isoenzymes, the GSH conjugation of BIU enantiomers was studied with human liver and intestinal cytosolic fractions as well as purified human class alpha (GSTA1-1, GSTA2-2), mu (GSTM1a-1a) and pi (GSTP1-1) GST isoenzymes. Stereoselective GSH conjugation of BIU enantiomers was observed for most human liver and intestinal cytosolic fraction. In general, the cytosolic fractions preferentially conjugated S-BIU. Stereoselective preference for GSH conjugation of S-BIU was also observed for GSTA2-2 and GSTM1a-1a, whereas GSTA1-1 was not selective for either of the BIU enantiomers. GSTP1-1 did not catalyse conjugation of R- and S-BIU. Quantification of the GST isoenzymes in the liver cytosolic fractions showed that the stereoselectivity towards S-BIU was related to the profile and amount of GST subunits in the cytosolic fractions. The discrepancy in stereoselectivity between the BIU pharmacokinetics in vivo and the GSH conjugation of BIU enantiomers in vitro is discussed. In addition, since in contrast to human GSTM1a-1a, rat class Mu isoenzymes prefer R-BIU, the present results indicate that related isoenzymes in different species may have a different stereoselectivity.

Bromisovalum↗