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PubMed1990

Effects of selected chemicals on the glutathione status in the male reproductive system of rats.

Previous studies have suggested a significant role for reproductive tract glutathione in protecting against chemical-induced germ-cell mutations. Therefore, a number of compounds were tested for their ability to perturb glutathione levels in the testes and epididymides as well as liver following single acute dosages to rats. Phorone (250 mg/kg), isophorone (500 mg/kg), and diethyl maleate (500 mg/kg) significantly reduced glutathione in the liver and in both reproductive organs examined. Methyl iodide (100 mg/kg), trimethyl phosphate (600 mg/kg), naphthalene (500 mg/kg), acetaminophen (1500 mg/kg), and pentachlorophenol (25 mg/kg) affected hepatic and epididymal glutathione, but had little or no effect on testicular levels. The ability of isophorone to enhance the covalent binding of tritiated ethyl methanesulfonate (3H-EMS) to spermatocytes was assessed. Perturbation of reproductive tract glutathione by isophorone treatment significantly enhanced the extent of 3H-EMS-induced binding to sperm heads. The temporal pattern of ethylations in sperm heads was consistent with the stage of sperm development known to be susceptible to ethylations by EMS. Therefore, chemical-induced lowering of glutathione in the male reproductive tract may be a mechanism for potentiation of chemical-induced germ-cell mutations.

Animals↗
PubMed1988

Application of pharmacokinetics to derive biological exposure indexes from threshold limit values.

The importance of incorporating the fundamental concepts of pharmacokinetics into biological monitoring program that involve the collection of various body fluid and tissue specimens is discussed. The application of these principles to establish biological exposures indexes bioequivalent to airborne exposure limits is described. Specific illustrative examples involving acetone, aniline, benzene, carbon tetrachloride, dieldrin, ethylbenzene, hexane, lead, methylene chloride, pentachlorophenol, phenol, styrene, toluene and xylene are presented.

Acetone↗
PubMed1992

Lactic acid secretion by human neutrophils. Evidence for an H+ + lactate- cotransport system.

The pathway by which L-lactate (Lac) crosses the plasma membrane of isolated human neutrophils was investigated. The influx of [14C]Lac from a 2 mM Lac, 145 mM Cl-, 5.6 mM glucose medium was approximately 1.5 meq/liter of cell water.min and was sensitive to the organomercurial agent mersalyl (apparent Ki approximately 20 microM), to alpha-cyano-4-hydroxycinnamate (CHC), the classical inhibitor of monocarboxylate transport in mitochondria, and to UK-5099 (apparent Ki approximately 40 microM), a more potent analogue of CHC. Transport was also strongly blocked (greater than 80%) by 1 mM of either 3,5-diiodosalicylic acid, MK-473 (an indanyloxyacetate derivative), or diphenyl-amine-2-carboxylate, and by 0.4 mM pentachlorophenol, but not by 1 mM ethacrynic acid, furosemide, or the disulfonic stilbenes SITS or H2DIDS. One-way [14C]Lac efflux from steady-state cells amounted to approximately 6 meq/liter.min and was likewise affected by the agents listed above. Influx, which was membrane potential insensitive and Na+ independent, displayed a strong pH dependence: extracellular acidification enhanced uptake while alkalinization inhibited the process (pK' approximately 5.7 at 2 mM external Lac). The rate of [14C]Lac influx was a saturable function of external Lac, the Km being approximately 7 mM. Steady-state cells exhibited an intracellular Lac content of approximately 5 mM and secreted lactic acid into the bathing medium a a rate of approximately 4 meq/liter.min. Secretion was completely suppressed by 1 mM mersalyl which inactivates the carrier, leading to an internal accumulation of Lac. That the Lac carrier truly mediates an H+ + Lac- cotransport (or formally equivalent Lac-/OH- exchange) was documented by pH-stat techniques wherein an alkalinization of poorly buffered medium could be detected upon the addition of Lac; these pH changes were sensitive to mersalyl. Thus, the Lac carrier of neutrophils possesses several features in common with other monocarboxylate transport systems in erythrocytes and epithelia.

Biological Transport↗
PubMed1989

4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate--a major metabolite of the food mutagen 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP) in the rat.

Isolated rat hepatocytes (4 X 10(6) cells/ml metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) (100 microM) to at least eight different metabolites in a 4 h duration. The major metabolite formed was 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP sulfate). Its rate of formation was increased in hepatocytes from PCB pretreated animals in comparison to hepatocytes from untreated animals. One of the other metabolites was the unconjugated derivative of the sulfate (4'-OH-PhIP). This metabolite was also found after in-vitro incubations of rat liver microsomes from PCB or beta-naphthoflavone pretreated animals. The evidence for the proposed structure of the major metabolite is based on [1H]NMR and UV spectroscopy, incorporation of radiolabelled sulfate and arylsulfatase-lability. The formation of 4'-PhIP sulfate was inhibited by the P-450 inhibitors alpha-naphthoflavone and metyrapone and when incubated in sulfate-free medium added the sulfotransferase inhibitor pentachlorophenol. 4'-PhIP sulfate was also the major metabolite of PhIP in the urine of exposed rat.

Animals↗
PubMed1990

Bioactivation of 2-nitrofluorene to reactive intermediates that bind covalently to DNA, RNA and protein in vitro and in vivo in the rat.

The activation of 2-nitrofluorene (2-NF) to reactive intermediates that bind covalently to DNA, RNA and protein has been investigated both in vitro and in the rat in vivo. In vitro, such binding was catalyzed by the hepatic microsomal fraction, was NADPH dependent and could be inhibited by SKF 525A, an inhibitor of cytochrome P450. The generation of reactive intermediates therefore is most likely catalyzed by cytochrome P450. Covalent binding of 2-NF could not be prevented by glutathione, N-acetylcysteine and other thiol-containing compounds. It could be partially prevented by guanosine, presumably because it traps the reactive intermediate(s). Under normal oxygenation conditions 2-NF was also covalently bound in freshly isolated hepatocytes; pretreatment of rats with an inducer of cytochrome P450, Aroclor 1254, gave rise to a higher rate of covalent binding in hepatocytes. Covalent binding of 2-NF to cellular macromolecules also occurred in vivo, both after oral and i.v. administration. Pentachlorophenol, a selective sulfation inhibitor, did not influence the covalent binding of 2-NF; therefore, the reactive intermediate is not formed by sulfation of N-hydroxy-2-acetylaminofluorene, which could be a metabolite of 2-NF. It is concluded that the reactive intermediates most likely can be formed from 2-NF by the cytochrome P450 enzyme system.

Animals↗
PubMed1990

Self-catalyzed irreversible inactivation of rat hepatic aryl sulfotransferase IV by N-hydroxy-2-acetylaminofluorene.

Rat hepatic aryl sulfotransferase IV catalyzes the sulfonation of the hepatocarcinogen, N-hydroxy-2-acetylaminofluorene. The resulting reactive N-O-sulfate ester is believed to be the ultimate carcinogenic species responsible for the induction of hepatic neoplasia. Previous studies have shown that dietary administration of either 2-acetylaminofluorene or N-hydroxy-2-acetylaminofluorene to rats is accompanied by a rapid decline in hepatic aryl sulfotransferase activity in vivo. In the present study, preincubation of purified rat hepatic aryl sulfotransferase IV with N-hydroxy-2-acetylaminofluorene resulted in rapid, time-dependent enzyme inactivation. This in vitro inactivation was not reversed by dialysis or gel filtration. Inclusion of excess nucleophile, methionine, resulted in considerable but not complete protection from inactivation. The inactivation was PAPS dependent and blocked by the sulfotransferase inhibitor, pentachlorophenol. The above observations and the apparent pseudo first-order kinetics observed suggest that the inactivation was in part mechanism based. Mechanism-based inactivation of the aryl sulfotransferases has not been previously reported. Furthermore, the results of the present study indicate that the previously reported in vivo decline in rat hepatic aryl sulfotransferase activity may be attributable in part to enzyme inactivation by its own reactive product.

2-Acetylaminofluorene↗
PubMed1991

Effect of rat liver cytosolic enzymes and cofactors on mutagenicity of 1-amino-8-nitropyrene.

1-Amino-8-nitropyrene (1,8-ANP), a product of 1,8-dinitropyrene metabolism by either bacterial or mammalian enzymes, is weakly mutagenic to the 'classical nitroreductase'-deficient Salmonella tester strain TA98NR. The addition to the test system of rat liver cytosol without cofactors did not produce any effect on the 1,8-ANP mutagenic response toward TA98NR strain. Conversely, when both rat hepatic cytosol and NADPH (1 mM) were added to the mutagenicity assay, a 10-fold increase in 1,8-ANP mutagenic activity was observed. This suggests the involvement of rat hepatic cytosolic NADPH-dependent nitroreductase(s) in 1,8-ANP mutagenic activation. The addition to the mutagenesis assay of pentachlorophenol, an inhibitor of O-acetyltransferase and sulfotransferase, produced a dose-dependent decrease of 1,8-ANP mutagenic activation, whereas 2,6-dichloro-4-nitrophenol, a more specific inhibitor of sulfotransferase than O-acetyltransferase, did not affect the activation of 1,8-ANP to a mutagen at concentrations that selectively inhibit only bacterial sulfotransferase. This indicates that bacterial O-acetyltransferase but not sulfotransferase plays a role in the mutagenic activation of 1,8-ANP. Addition of acetyl co-enzyme A (AcCoA) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), cofactors for O-acetyl-transferase and sulfotransferase respectively, to the test system caused a dose-dependent inhibition of 1,8-ANP mutagenic activation by rat liver cytosol and NADPH, probably due to the formation of highly reactive O-acetoxy and N-sulfate ester derivatives of 1,8-ANP, which react with nucleophilic sites before reaching bacterial DNA. This hypothesis was confirmed by DNA covalent binding in in vitro experiments showing that both the cofactors AcCoA and PAPS enhanced the NADPH/rat liver cytosol-mediated covalent binding of 1,8-ANP to DNA from calf thymus 10- and 3-fold respectively. It seems likely that rat hepatic cytosolic nitroreductases activate 1,8-ANP to an N-hydroxyarylamine derivative which can be further metabolized to mutagenic species by either bacterial or mammalian O-acetyltransferase.

Acetyl Coenzyme A↗
PubMed1992

Reaction product inactivation of aryl sulfotransferase IV following electrophilic substitution by the sulfuric acid ester of N-hydroxy-2-acetylaminofluorene.

Rat liver N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) sulfotransferase activity is mediated by aryl sulfotransferase IV (AST IV) and causes the bioactivation of N-OH-2AAF to a highly reactive sulfuric acid ester form putatively capable of inducing liver cancer. Dietary administration of 2-acetylaminofluorene (2AAF) to induce hepatocarcinogenesis in rats has been shown to cause a rapid loss in N-OH-2AAF sulfotransferase activity. A possible mechanism for the in vivo loss in sulfotransferase activity may be the PAPS-dependent, sulfotransferase-catalyzed, reaction product inactivation of the enzyme by covalent reaction with the N-OH-2AAF sulfuric acid ester. In vitro studies to evaluate this possibility utilized a highly purified form of AST IV and measured the extent of PAPS-dependent interaction between the enzyme and N-OH-2[9-14C]AAF. The results showed the presence of a adenosine-3'-phospho-5'-phosphosulfate (PAPS)-dependent 14C-labeling of AST IV. The labeling could be blocked if the sulfotransferase inhibitor pentachlorophenol was present. Analysis of 14C-labeled AST IV following alkaline digestion and chromatography of digestion products indicated that AST IV cysteine and methionine residues were primary sites of 2[9-14C]AAF adduction. Studies involving the pretreatment of AST IV with PAPS and N-OH-2AAF prior to the measurement of N-OH-2AAF sulfotransferase activity showed a close parallel between formation of the AST IV cysteine-2AAF adduct and loss of activity. Similar studies showed that enzyme inactivation and cysteine-2AAF adduct formation could be blocked when excessive amounts of a competing nucleophile, methionine, were present during the pretreatment step, suggesting that inactivation does not proceed by a mechanism-based process. Finally, experiments involving prior reaction of AST IV with the thiol-blocking agent, N-ethylmaleimide, before measurement of enzyme activity showed essentially full loss of sulfotransferase activity and suggested that formation of AST IV cysteine-2AAF adducts could be a mechanism for enzyme inactivation. These results indicate that the in vitro inactivation of AST IV by the reactive N-OH-2AAF sulfuric acid ester is accompanied by covalent binding to AST IV, possibly through the formation of cysteine-2AAF adducts, and suggests that this mechanism merits further consideration as a basis for the loss of N-OH-2AAF sulfotransferase activity in vivo.

Animals↗
PubMed1992

Participation of rat liver cytochrome P450 2E1 in the activation of N-nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009).

The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.

Acetyltransferases↗
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↗
PubMed1995

Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague-Dawley rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in fried beef, is mammary gland carcinogen in rats. Using the 32P-postlabeling method, PhIP-DNA adduct levels were measured in mammary epithelial cells isolated from female Sprague-Dawley rats given 10 daily doses of PhIP (75 mg/kg, p.o.) according to a protocol previously shown to induce mammary gland cancer. At 24 h, 48 h, 1 week and 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SD] were 10.2 +/- 0.7, 7.9 +/- 2.7, 2.2 +/- 0.6 and 0.9 +/- 0.03 respectively. When isolated rat mammary epithelial cells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP (45 microM, 1 h, 37 degrees C), PhIP-DNA adducts were detected in cell DNA (RAL = approximately 97 x 10(7); however, no adducts were detected in cells incubated with PhIP (200 microM, 15 h, 37 degrees C). Incubating cells with pentachlorophenol, an inhibitor of acetyltransferase, or incubating cells at 0-4 degrees C, reduced N-hydroxy-PhIP adduct levels by 45 and 75% respectively, indicating that formation of N-hydroxy-PhIP adducts was largely due to metabolic activation. Further studies showed that rat mammary gland microsomes had little capacity to N-hydroxylate PhIP, as assayed by the mutagenic activation of PhIP in the Ames Salmonella assay. In contrast, N-hydroxy-PhIP was metabolically activated by cytosol-catalyzed PhIP-DNA adduct formation to calf thymus DNA incubated in vitro with N-hydroxy-PhIP (2 microM) in the presence of acetyl CoA. Notably, mammary cytosolic O-acetyltransferase activation of N-hydroxy-IQ or N-hydroxy-MeIQx. All three N-hydroxylamines were activated via cytosolic proline aminoacyl-tRNA synthetase and phosphorylase, although the activities of these enzymes were approximately 100-fold lower than O-acetyltransferase. No mammary cytosolic sulfotransferase activation could be detected with any of the N-hydroxylamines. Our results are consistent with the notion that PhIP-DNA adduct formation and initiation of carcinogenesis in the rat mammary gland may be associated with N-hydroxylation of PhIP outside the mammary gland, transport of the N-hydroxylamine to the mammary gland and subsequent in situ O-acetyltransferase-catalyzed activation of N-hydroxy-PhIP.

Acetyltransferases↗
PubMed2001

Induction of direct adducts, apurinic/apyrimidinic sites and oxidized bases in nuclear DNA of human HeLa S3 tumor cells by tetrachlorohydroquinone.

DNA damage induced by tetrachlorohydroquinone (Cl(4)HQ), the quinonoid metabolite of pentachlorophenol (PCP), was investigated in human HeLa S3 tumor cells. Formation of one major and two minor DNA adducts in cells treated with Cl(4)HQ (50-300 microM) was detected by (32)P-post-labeling assay and the adducts accumulated over the course of the experiment (0.5-2 h), with total adduct levels estimated to be 3-6 per 10(8) nucleotides. These adducts did not correspond to those derived from calf thymus DNA treated with tetrachloro-1,4-benzoquinone. Results from the apurinic/apyrimidinic (AP) sites assay indicated that the number of AP sites was 2-fold greater in cells exposed to Cl(4)HQ (300 microM) than the corresponding control. Further characterization of the AP sites confirmed that Cl(4)HQ induced predominantly (75%) putrescine-excisable AP sites in HeLa S3 cells. In parallel, the concentration of 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in cells treated with Cl(4)HQ for 0.5 and 2 h was increased 2- and 5-fold, respectively, compared with the control. The extent of oxidative DNA damage induced by Cl(4)HQ was approximately two orders of magnitude greater than those of direct DNA adducts. Overall, it appears that reactive oxygen species mediate the parallel formation of AP sites and 8-HO-dG in HeLa S3 cells following treatment with Cl(4)HQ and that the contribution of depurination/depyrimidination of direct DNA adducts is relatively insignificant compared with the formation of oxidized AP sites. We conclude that putrescine-excisable AP sites represent a major type of ROS-mediated oxidative DNA damage in cellular DNA induced by Cl(4)HQ and may play a role in PCP-induced clastogenicity in mammalian cells.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1985

N-sulfoöxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene in the livers of infant male C57BL/6J x C3H/HeJ F1 (B6C3F1) mice.

The hepatic DNA of 12-day-old male B6C3F1 (C57BL/6J X C3H/HeJ) mice given an i.p. dose of 0.06 or 0.11 mumol/g body weight of N-hydroxy-[3H]-2-acetylaminofluorene (N-hydroxy-AAF) contained at 9 h approximately 3 or 6 pmol of N-(deoxyguanosin-8-yl)-2-aminofluorene adducts per mg. Together the level of the two acetylated adducts N-(deoxyguanosin-8-yl)-2-acetylaminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene was less than or equal to 10% of this amount. The same doses of unlabeled carcinogen induced by 10 months a 100% incidence of hepatomas with averages of 10 and 15 hepatomas per mouse, respectively. Injection of 0.04 mumol/g body weight of pentachlorophenol (PCP) 45 min before the dose of N-hydroxy-AAF decreased the number of adducts in the DNA by 90% and the average number of hepatomas per liver by 80-90%. As compared to their normal male littermates, male brachymorphic B6C3F2 mice, which are deficient in hepatic 3'-phosphoadenosine-5'-phosphosulfate (PAPS), treated with N-hydroxy-AAF formed only 25% as many hepatic DNA adducts and developed only 10% as many hepatomas. Hepatic cytosols from 12-day-old B6C3F1 mice contained PAPS-dependent sulfotransferase activity for N-hydroxy-2-aminofluorene (N-hydroxy-AF), a previously unrecognized activity, as well as sulfotransferase activity for N-hydroxy-AAF; both activities were inhibited 60% by 1 microM and greater than or equal to 80% by 10 microM PCP. Cytosolic acetyl coenzyme A-dependent acetyltransferase activity for N-hydroxy-AF, cytosolic N,O-acyltransferase activity for N-hydroxy-AAF, and microsomal deacetylase for N-hydroxy-AAF were not significantly inhibited by PCP under these conditions. The above data strongly indicate that N-sulfoöxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-AAF in the livers of infant male B6C3F1 mice.

2-Acetylaminofluorene↗
PubMed1985

Elevation of hepatic microsomal epoxide hydrolase activity by 2-acetylaminofluorene: role of metabolism.

Brief exposure of rats to hepatocarcinogenic agents causes a rapid elevation in hepatic microsomal epoxide hydrolase (EH) activity. Previous studies have demonstrated that animals which are resistant to the hepatocarcinogenic effects of 2-acetylaminofluorene (AAF) are also resistant to EH induction by this compound. The studies described here were designed to examine the role of several individual metabolic pathways on the induction of EH by AAF. EH was increased 4-fold in male Fischer 344 (F-344) or Sprague-Dawley (SD) rats treated with AAF; in female rats, deficient in N-hydroxylase and sulfotransferase activities, the activity was increased only 2-fold. Pretreatment of male F-344 rats with inducers of cytochrome P-448 activity caused a reduction in the EH response to AAF, probably due to a greater increase in ring-hydroxylation than N-hydroxylation of the AAF. Although AAF elicited only a small EH elevation in female F-344 rats, N-hydroxy-2-acetylaminofluorene (N-OH-AAF) caused a large increase in these animals. The N-OH-AAF-induced increase was partially blocked by pretreatment with pentachlorophenol, an inhibitor of sulfotransferase activity, in both male and female F-344s. In female SD rats, possessing minimal sulfotransferase activity, N-OH-AAF treatment caused only a slight elevation of EH activity. Pretreatment of male F-344 rats with inhibitors of deacetylase activity had no effect on N-OH-AAF-dependent EH induction. These observations are consistent with the suggestion that formation of the sulfate conjugate of N-OH-AAF is necessary for elevation of EH by this compound.

2-Acetylaminofluorene↗
PubMed1987

Covalent binding to DNA and mutagenicity of 2,4-diaminotoluene metabolites produced by isolated hepatocytes and 9000 g supernatant from Fischer 344 rats.

Primary cultures of hepatocytes from adult male F344 rats were used to investigate the activation of the hepatocarcinogen 2,4-diaminotoluene (2,4-DAT) to metabolites which bound covalently to DNA. Covalent binding of 2,4-DAT to DNA was significantly greater than that of a non-carcinogenic isomer, 2,6-DAT. Treatment of male rats with 5,6-benzoflavone (BNF), an inducer of cytochrome P-450c and P-450d, had no effect on the binding of 2,4-DAT to DNA of hepatocytes from these animals. However, treatment of hepatocytes in vitro with metyrapone or piperonyl butoxide, two general inhibitors of P-450 enzymes, inhibited the binding of 2,4-DAT to DNA by approximately 80-85%. Two inhibitors of sulfation, pentachlorophenol and 2,5-dichloro-4-nitrophenol, also inhibited DNA binding in hepatocytes from both BNF-induced (91 and 85% respectively) and control rats (82 and 41% respectively), indicating that sulfation may also be required. 2,4-DAT was a more potent mutagen than 2,5- or 2,6-DAT in the Ames Salmonella mutagenesis assay using hepatic S9 fractions from F344 rats as an activating system. In contrast to DNA binding, activation of 2,4-DAT to mutagens by S9 fractions from BNF-treated rats was greater than that by S9 from control rats. The present study shows that 2,4-DAT is activated by hepatocytes of F344 rats to products which bind covalently to DNA. Both cytochrome P-450 and sulfation appear to be involved in the activation.

Animals↗
PubMed1987

Sulphotransferase-mediated covalent binding of the carcinogen 7,12-dihydroxymethylbenz[a]anthracene to calf thymus DNA and its inhibition by glutathione transferase.

The carcinogen, 7,12-dihydroxymethylbenz[a]anthracene (DHBA), bound covalently through its 7-methylene carbon to calf thymus DNA via DHBA 7-sulphate, a regiospecifically formed, reactive metabolite, when incubated with rat liver cytosol in the presence of 3'-phosphoadenosine 5'-phosphosulphate (PAPS). The hydroxysteroid sulphotransferase inhibitor, dehydroepiandrosterone sulphate, strongly retarded the covalent binding of DHBA to DNA as well as the DHBA 7-sulphate formation from DHBA in the biological system, while pentachlorophenol and dichloronitrophenol showed little effect on these reactions. DHBA 7-sulphate was a good substrate for rat liver cytosolic glutathione (GSH) transferases, so that the PAPS-dependent covalent binding of DHBA to DNA could be markedly retarded in the presence of GSH with concomitant formation of a significant amount of the stable conjugate, S-(12-hydroxymethylbenz[a]anthracen-7-yl)methylglutathione. From DNA, incubated with DHBA in the presence of the hepatic cytosol and PAPS as well as with DHBA 7-sulphate alone, two purine base adducts were isolated after hydrolysis. The purine base adducts accounted for 70% of the total covalent binding of the carcinogen or its 7-sulphate to the nucleic acid and were identified with synthetic specimens as N6-(12-hydroxymethylbenz[a]anthracen-7-yl)-methyladenine and N2-12-hydroxymethylbenz[a]anthracen-7-yl)-methylguanine. The ratio of the adenine to guanine adducts was 1:2.5 with DNA, incubated with DHBA in the presence of hepatic cytosol and PAPS as well as with various concentrations of DHBA 7-sulphate.

Animals↗
PubMed2003

Use of biosensors to screen urine samples for potentially toxic chemicals.

Forensic toxicology laboratories are often required to implicate or exclude poisoning as a factor in a death or unexplained illness. An analytical tool which enables toxicologists to screen a wide variety of common poisons would be extremely useful. In this paper, we describe the use of a bacterial biosensor for detecting the presence of commonly encountered potentially toxic chemicals in urine. The biosensor responds to any chemical that causes metabolic stress to the bacterial cell and the response is in direct proportion to the concentration of the stressor. This allows a measure of the concentration of a toxicant in urine, without knowing exactly what the toxic compound(s) may be. This affords a distinct advantage over conventional analytical techniques, which require an extensive screening program before it is even known that a toxic compound is present. This preliminary investigation has shown that this biosensor can indicate the presence, in urine, of herbicides such as glyphosate, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid; the biocide pentachlorophenol; or inorganic poisons such as arsenic, mercury, and cyanide. The biosensor was also shown to be sensitive to a concentration range of these toxicants likely to be found in samples submitted for toxicological analysis.

Biosensing Techniques↗
PubMed1995

Fish oil enhances pentachlorobenzene metabolism and reduces its accumulation in rats.

To investigate the influence of dietary fats on the metabolism and excretion of pentachlorobenzene (PECB), rats were fed semipurified diets containing soybean oil, lard or fish oil (10 g/100 g diet) for 2 wk. Rats then received a single dose of PECB by intragastric gavage. Blood concentration of pentachlorophenol (PCP), a major metabolite of PECB, was higher after 5 h through d 3 and that of PECB was lower after d 5 in the rats fed fish oil than in rats fed the other fats. After d 5, the highest ratio of PCP/PECB in the liver was seen in the fish oil-fed rats. Furthermore, fish oil intake resulted in a markedly lower concentration of PECB in fat tissues, where a large amount of PECB is typically distributed due to its lipophilic property. These findings indicate that fish oil feeding enhances PECB metabolism, thereby decreasing PECB residues in the body. Fish oil-fed rats had a smaller epididymal fat tissue mass compared with lard- or soybean oil-fed rats. The small mass of the fat tissues apparently limited the accumulation of PECB in those tissues and thus accelerated PECB metabolism in the liver.

Adipose Tissue↗