Organ-specific DNA damage of tris(2,3-dibromopropyl)-phosphate and its diester metabolite in the rat.

The organ specificity of tris(2,3-dibromopropyl)phosphate(Tris-BP)-induced DNA damage was investigated in the rat 2 h after a single i.p. injection of 350 mumol/kg. Extensive DNA damage, measured with the alkaline elution method, was found in the kidney, liver and small intestine. Less, but significant DNA damage was detected in the brain, lung, spleen, large intestine and testis. The role of different pathways in the activation of Tris-BP to DNA damaging products was studied in isolated liver and testicular cells. Concentrations as low as 2.5-5 microM Tris-BP caused DNA damage in the hepatocytes, whereas an approximately 10-fold higher concentration was needed in testicular cells to produce a similar amount of DNA damage. Depletion of GSH by diethyl maleate (DEM) did not affect the extent of DNA damage caused by Tris-BP in the liver cells, but blocked the genotoxic effect in testicular cells. Two specifically deuterated Tris-BP analogs, C3D2-Tris-BP and C2D1-Tris-BP, were significantly less potent in causing DNA damage than the protio compound in isolated liver cells and were somewhat less potent in testicular cells. The major urinary metabolite of Tris-BP, bis(2,3-dibromopropyl)phosphate (Bis-BP), was less potent than Tris-BP in causing kidney DNA damage after in vivo exposure. Furthermore, Bis-BP induced substantially less DNA damage in isolated liver and testicular cells. Similar to the effect of DEM on the DNA damage caused by Tris-BP, the DNA damage caused by Bis-BP could be decreased by DEM-pretreatment in testicular cells but not in liver cells. The present study shows that Tris-BP is a potent multiorgan genotoxic agent in vivo. The in vitro data indicate that P-450 mediated metabolism of Tris-BP is more important than activation by glutathione S-transferases of Tris-BP in liver cells, whereas the latter activation pathway seems to be most important in testicular cells.

Genotoxic effects of 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ) in rats measured by alkaline elution.

The alkaline elution method was used to examine genotoxic effects of MeIQ in various organs of rats after in vivo exposure. No DNA damage could be observed in the stomach, small and large intestine, liver, kidney or testis of male Wistar rats 2 h after a single intraperitoneal dose of 80 mg/kg MeIQ. In rats that had been pretreated with Aroclor 1254 (PCB), MeIQ induced significant DNA damage in the liver after both oral and intraperitoneal injection. MeIQ induced DNA damage in the large intestine, liver and kidney of male F344 rats given a single intraperitoneal dose of 80 mg MeIQ/kg or fed 0.03% MeIQ for 13 days. The DNA damage did not seem to accumulate during the feeding period.

Toxic effects of cyclophosphamide in differentiating chicken limb bud cell culture using rat liver 9,000 g supernatant or rat liver cells as an activation system: an in vitro short-term test for proteratogens.

Cells from 4-day chicken embryo limb buds plated as micromass cultures differentiate and form cartilage nodules after a 5- to 6-day growth period. The innate ability of these cells to biotransform compounds, such as cyclophosphamide (CP), into reactive metabolites is apparently inadequate. Protocols used rat liver S9 from Aroclor 1254-pretreated (PCB) rats or hepatocytes from control rats or polychlorinated biphenyls (PCB)-pretreated rats and were added to micromass cultures with CP causing concentration-related toxicity in the cell cultures. Coculturing chicken limb bud cells with PCB-hepatocytes was the most efficient method, yielding an IC50 of 2 micrograms CP per ml. Toxic CP metabolites accumulated in the medium of PCB-hepatocyte cultures and were quite stable. The toxicity of bioactivated CP was nearly identical for both proliferation and cartilage proteoglycan accumulation.

Comparative genotoxicities of procarbazine and two deuterated analogs in mammalian cells in vitro and in vivo.

N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine; 50-1000 micrograms/ml) induced DNA damage in hepatocytes measured by an automated alkaline elution method, whereas no significant increase in unscheduled DNA synthesis was seen. In hepatocytes isolated from PCB-treated rats, DNA damage was detected in both test systems at concentrations as low as 1-10 micrograms/ml. DNA damage, as measured by alkaline elution and sister-chromatid exchange(s), was observed also in V79 cells incubated with PCB-hepatocytes. In contrast, no mutagenic activity was observed in the Salmonella typhimurium strain TA1530 co-incubated with the hepatocytes. Exposure of rats to low doses of procarbazine (25-50 mg/kg) caused DNA damage measured by alkaline elution in liver and testis, with the liver being somewhat more sensitive. The genotoxicity caused by procarbazine was increased by a factor of 2-3 in both organs by PCB-treatment of the rats. N-isopropyl-alpha-(2-methyl-hydrazino)-p-[alpha,alpha-2H2]toluamide (d2-procarbazine), was found to cause significantly less genotoxicity in control rats than either procarbazine itself, or N-isopropyl-alpha-(2-[alpha,alpha,alpha-2H3]methylhydrazino)-p-tol uamide (d3-procarbazine). This indicates that benzylic C-H oxidation of procarbazine is an important step in the activation of procarbazine to genotoxic metabolites in uninduced rats.

Different mechanisms are involved in DNA damage, bacterial mutagenicity and cytotoxicity induced by 1,2-dibromo-3-chloropropane in suspensions of rat liver cells.

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measured by an automated alkaline elution method, in suspensions of rat liver parenchymal cells at low concentrations (1-10 microM). At much higher concentrations (0.5-2.5 mM), DBCP was metabolized to products that were mutagenic to Salmonella typhimurium TA100 co-incubated with the liver cells. At these higher concentrations a marked depletion of cellular glutathione was seen and at 2.5 mM DBCP was cytotoxic. Perdeuterated DBCP (D5-DBCP) caused less DNA damage in the liver cells than DBCP, most likely because of decrease in cytochrome P-450 dependent metabolism. A more pronounced decrease in mutagenicity occurred with D5-DBCP compared to DBCP, whereas the two compounds were equally cytotoxic. Preincubation of the liver cells with diethylmaleate or buthionine sulfoximine, to lower cellular levels of glutathione, decreased DBCP induced DNA damage. The decrease in DNA damage was proportional to the decrease in cellular glutathione levels. In contrast, diethylmaleate enhanced DBCP-induced bacterial mutagenicity and cellular cytotoxicity. The cytotoxic effect could be partly blocked by addition of ascorbate. From the data presented we suggest that: (i) cytochrome P-450 dependent oxidation as well as glutathione conjugation are involved in DBCP induced DNA damage, (ii) cytochrome P-450 dependent oxidation leads to formation of products mutagenic to bacteria and (iii) the cytotoxicity induced by DBCP in the liver cells in vitro is caused by oxidative damage following glutathione depletion and/or direct membrane damage.

Modulation of the mutagenic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in bacteria with rat-liver 9000 x g supernatant or monolayers of rat hepatocytes as an activation system.

An in vitro protocol was designed to separate the process of metabolic activation from the mutational events. Cultured rat hepatocytes were first incubated with the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). After the incubation period the medium was removed and further incubated with Salmonella typhimurium TA98. A high direct mutagenic activity of the culture medium was then measured. The half-lives of the mutagenic metabolites formed from IQ and MeIQ were in the order of 45 min. The presence of the cytochrome P450 inhibitors alpha-naphthoflavone and metyrapone during the pre-incubation period reduced the accumulation of mutagenic metabolites. No effects of ascorbate on the mutagenic effects of IQ and MeIQ were seen. (+)-Catechin, another antioxidant and free-radical scavenger, markedly enhanced the number of IQ/MeIQ-induced revertants when added to the hepatocytes. In contrast, (+)-catechin clearly decreased the number of revertants when 9000 X g supernatant from rat liver (S9) was used as an activation system. No marked effect of pentachlorophenol, an inhibitor of hepatocyte sulfation and bacterial O-acetylation, was seen using hepatocytes as an activation system, while the mutagenic activity of both IQ and MeIQ was reduced by 90% in the S9/Salmonella system. The addition of an inhibitor of glucuronidation, galactosamine, or the nucleophile glutathione caused no or only minor decreases in the genotoxic effects of the IQ compounds. With both S9 and hepatocytes as activation systems the relative mutagenic effects observed in the S. typhimurium strains TA98 and TA98 NR were in the same order of magnitude, while a large decrease was seen with TA98/1,8-DNP6. The results show that this in vitro test protocol may be useful as a tool to study mechanisms involved in the formation of mutagenic metabolites.

Metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline IQ and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in suspensions of isolated rat-liver cells.

Suspensions of rat-liver cells metabolized (14)C-labelled 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to metabolites that were ethyl acetate extractable, water soluble or covalently bound to macromolecules. The major ethyl acetate extractable metabolite(s) had a retention time on the high-performance liquid chromatograph (HPLC), an ultraviolet spectrum and an R(f) value on the thin-layer chromatograph that corresponded to those of the N-acetylated derivative. Relatively more of this metabolite was formed from MeIQ than from IQ. In contrast, IQ was more easily converted to water-soluble metabolites than was MeIQ. The amount of covalently bound metabolite(s) found with MeIQ as test substance was larger than that found with IQ. No major increase in the ethyl acetate-extractable metabolites was obtained after incubation of the aqueous phase with beta-glucuronidase or aryl sulphatase in comparison with untreated controls. HPLC analysis showed that after acid hydrolysis of the water-soluble metabolites, about 55 and 20% of the hydrolysed metabolites had retention times that were the same as those of IQ and MeIQ, respectively. The ratio between covalently bound (activated) metabolites and water-soluble (detoxified) metabolites was larger for MeIQ than for IQ. From these data, one would expect MeIQ to be more potent than IQ as a liver carcinogen in male rats.

Modulation of cytotoxic and genotoxic effects of 2-acetylaminofluorene in rat and hamster hepatocytes by 3-methylcholanthrene pre-treatment.

It is well known that 2-acetylaminofluorene (AAF)-induced liver cancer is reduced by simultaneous administration of 3-methylcholanthrene (MC) in the rat, but not in the hamster. The present report examines the effects of MC pre-treatment on the metabolism and toxicity of AAF in monolayer cultures of hepatocytes. Hepatocytes isolated from pre-treated animals of both species metabolized AAF and 2-aminofluorene (AF) to metabolites mutagenic to Salmonella typhimurium more efficiently than hepatocytes from control animals. MC-pre-treated rat hepatocytes showed increased responses to AAF- and AF-induced unscheduled DNA synthesis, while MC-pre-treated hamster hepatocytes were less responsive than the untreated hepatocytes. Increased cytotoxic effects of AAF were observed in MC-pre-treated rat hepatocytes, whereas AAF was not cytotoxic in hamster hepatocytes from either pre-treated or control animals. MC pre-treatment caused increased rates of formation of C-hydroxylated, N-hydroxylated, water-soluble and covalently macromolecular bound AAF metabolites in both species. No significant effect of MC pre-treatment was seen on the formation of AF from AAF. A large decrease in the ratio between covalently macromolecular bound (activated) metabolites and the sum of C-hydroxylated and water-soluble (detoxified) AAF metabolites, was seen after MC pre-treatment of rat hepatocytes, whereas no or only a minor decrease was observed in hamster hepatocytes. This ratio correlated much better with the in vivo carcinogenicity data than the other parameters such as mutagenicity, DNA repair or covalent macromolecular binding. Thus, the hypothesis that AAF-induced liver cancer depends less on the rate at which AAF is activated, but more on the relative proportion of the dose which is activated, is supported by the present data.

Species differences in the metabolism of 2-acetylaminofluorene by hepatocytes in primary monolayer culture.

Monolayers of hepatocytes from mouse, hamster, rat, and guinea pig metabolized 2-acetylaminofluorene (AAF) to ether-extractable, water-soluble as well as covalently macromolecular bound products. Hamster hepatocytes showed the highest rate of formation of ether-extractable metabolites, rat and guinea pig the lowest. These species differences reflected mainly differences in the formation of 2-aminofluorene, the dominating ether-extractable metabolite formed. Detectable levels of N-hydroxy-AAF (greater than 1 nmol/10(6) cells) were only obtained with hamster hepatocytes. The major C-hydroxylated metabolites in the species tested were 7- and 9-hydroxy-AAF. Hepatocytes from guinea pig and hamster showed the highest rate of formation of C-hydroxylated and water-soluble metabolites, rat hepatocytes the lowest. The highest rate of covalent macromolecular binding by AAF metabolites was found with hamster hepatocytes, followed by hepatocytes from rat, guinea pig, and mouse. The balance between activation and detoxification reactions of AAF in hepatocytes may be expressed as the ratio between covalently bound metabolites and the sum of C-hydroxylated and stable water-soluble metabolites. This ratio was far greater in rat hepatocytes followed by hamster, guinea pig, and mouse, and it correlated better with the species susceptibility to liver cancer than covalent binding as such. Thus, AAF-induced liver cancer may depend more on the relative degree of activation versus detoxification of the administered dose than on the absolute capacity of the liver to activate the carcinogen.