Brita Beije

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. activation by the isolated perfused rat liver

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.

On the Mechanism of the Hepatocarcinogenicity of Peroxisome Proliferators

The absence of a genotoxic action in the rat of several peroxisome proliferators (PP) has been confirmed by measuring gross degradation, unscheduled DNA-synthesis (UDS), as well as by measurement of single strand breaks using alkali unwinding in absence and presence of inhibitors of DNA-repair. Similar results were obtained even after drastically lowering the glutathione content of liver. Further, after oral administration of ciprofibrate, no potentiating effect was found in vivo on the generation of micronuclei in hepatocytes by ionizing radiation. The metabolically inert PP, perfluorooctanoic acid, was found to act as a promoter of liver tumors in the rat induced by diethylnitrosamine in an initiation-selection-promotion protocol. The results are discussed in light of available information concerning the mechanism of action of PPs.

Mutagenicity testing on Chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermanns system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.

Isolated liver perfusion--a tool in mutagenicity testing for the evaluation of carcinogens.

An isolated liver perfusion system suitable for the combination with Chinese hamster V79 cells is described. With this system, it is possible to study, with the V79 cells as genetic targets, the mutagenic effect of a chemical after metabolic activation in the intact organ. Those substances commonly used in mutagenicity testing as inducers of drug metabolising enzymes, i.e. Arochlor 1254. Phenobarbital(PB) and 3-Methylcholantrene(3-MC), were studied for their effect in the isolated perfused liver. PB increased the bile flow, which was not significantly affected by the other inducers. Only Arochlor caused a significant increase in the amino acid incorporation into plasma proteins and total liver proteins (expressed per mg liver protein). None of the inducers had an effect on gluconeogenesis from lactate or urea synthesis. All three inducers caused an increase in the level of microsomal P-450 enzymes, the biggest increase being seen after Arochlor-induction (170%), followed by PB(90%) and 3-MC(50%). Arochlor- and PB-induction had a dramatic effect on N- and C-oxygenation of N, N-dimethylaniline: N-oxygenation was decreased by 35% and 40% respectively and C-oxygenation increased by 130% and 140% respectively. The advantages of the isolated perfused liver as an intact metabolising unit is discussed in relation to other mutagenicity assays, in which subcellular fractions are used as the metabolising system.

Investigation of styrene in the liver perfusion/cell culture system. No indication of styrene-7,8-oxide as the principal mutagenic metabolite produced by the intact rat liver

Mutagenic effect of styrene and styrene-7,8-oxide was studied with the isolated perfused rat liver as metabolizing system and Chinese hamster V79 cells as genetic target cells. Styrene-7,8-oxide which is mutagenic per se was rapidly metabolized by the perfused rat liver. Thus no mutagenic effect was detected neither in the perfusion medium nor in the bile. However when styrene was added to the perfusion system, an increase in V79 mutants was observed regardless of where in the circulating perfusion medium the V79 cells were placed: the same effect was obtained with V79 cells close to the liver as well as at a distance from the liver. No mutagenic effect was observed in the bile. Simultaneous analysis of the styrene-7,8-oxide concentration in the perfusion medium, suggest that this metabolite is not the cause of the mutagenic effect observed during perfusion with styrene. The effect of the two test compounds on some liver functions was also studied. Both styrene and styrene-7,8-oxide changed the bile flow without affecting bile acid secretion: styrene caused a reduction in bile flow as compared to control perfusions and styrene-7,8-oxide increased the bile flow. Styrene, but not styrene-7,8-oxide, reduced gluconeogenesis from lactate. Styrene had no effect on the livers capacity to incorporate amino acids into plasma proteins, whereas styrene-7,8-oxide reduced the amino acid incorporation. The microsomal cytochrome P-450 content was not affected by the two test compounds. No alteration in microsomal N- and C-oxygenation of N,N-dimethylaniline (DMA) was observed with styrene-7,8-oxide or the lower styrene dose used (240 mumol), whereas the higher styrene concentration (480 mumol) reduced N-oxygenation and thus also the total DNA metabolism. It is suggested that the results on styrene and styrene-7,8-oxide found here using the liver perfusion/cell culture system mimic the metabolism expected to be found in the intact animal, thus indicating that styrene-7,8-oxide is not the principal mutagenic metabolite of styrene in vivo.

Induction of unscheduled DNA synthesis in liver and micronucleus in bone marrow of rats exposed in vivo to the benzidine-derived azo dye, Direct Black 38

The genotoxic activity of the benzidine-derived azo dye, Direct Black 38 (DB38), was studied in vivo, using two different genetic end-points: unscheduled DNA synthesis in liver (UDS) and bone marrow micronucleus (MN). Exposure times were 12, 24 or 36 h. Both assays were performed in the same rat, except for the 24-h exposure when only MN was investigated. For the liver UDS assay, the rat hepatocarcinogen, 6-dimethylaminophenylazobenzthiazole (6BT), was used as positive control and for the MN assay, cyclophosphamide (CP). In agreement with earlier results, 6BT gave rise to a dose-related increase in liver UDS after 12-h exposure to 25 or 50 mg/kg bw. After 36-h exposure, there was still an indication of a weak dose-response effect between 0 and 5 net nuclear grains (NG). DB38 induced liver UDS at the higher dose levels used (500 and 1000 mg/kg), and after both 12- and 36-h exposure. With the longer exposure time, a weak induction of UDS was also observed at 100 mg/kg. The strongest UDS induction (12.2 NG), was obtained in one rat after 36-h exposure to 500 mg/kg. DB38 also had a weak effect on the MN induction, which was statistically significant at the higher concentrations used. A dose-related response was observed at all exposure times used.

Oxidation and protein binding of aromatic amines by rat liver microsomes

Abstract The binding of DMA and AF to protein by rat liver microsomes was studied under various conditions and compared with the utilization of DMA for oxidative demethylation and N-oxide formation. According to current evidence only the former reaction involves cytochrome P-450 as oxygen activator. 1. (1)|When the binding was tested with detoxication inhibitors (CO, SKF 525-A, mercurials) known to differentiate between demethylation and N-oxidation, it gave an intermediate or biphasic response. A similar result was obtained after graded, structural disorganization of the membranes by sonication or cholate treatment. While a major part of the binding approximated the demethylation in its susceptibility to inhibition, a minor, less well-defined part was more resistant. On the basis of the difference in CO susceptibility, the former type of binding was considered cytochrome-dependent, while the latter may include cytochrome-independent components. 2. (2)|The binding of DMA was not in all respects a reflection of the demethylation or N-oxide formation. For instance, the binding of DMA was not inhibited (but in fact stimulated) at high substrate concentrations or after pretreatment of the microsomes with moderate concentrations of trypsin, while the demethylation was markedly reduced under these conditions. Similarly, the binding was not inhibited in microsomes after preincubation in plain medium, although the N-oxide formation greatly diminished after this treatment. These and other data suggest that the binding was related to intermediates in the oxygenation reactions, rather than to end products. The possibility is discussed that cytochrome-dependent and cytochrome-independent binding are related to the enzyme-substrate complexes involved in C- and N-oxygenation, respectively. Anomalous decomposition of these complexes may lead to a disconnection of bound reaction intermediates in the form of free radicals. 3. (3)|Under the conditions studied, the binding of AF was closely correlated with that of DMA.

Influence of cystein deficiency on the inhibition of hepatic microsomal detoxication by methyl mercury in two rat strains

Two rat strains, Wistar, strain R and Sprague--Dawley, were subjected to cystein deficiency and methyl mercury pretreatment, both separately and in combination, after which the hepatic microsomal N- and C-oxygenation of N,N-dimethylaniline (DMA) was studied. Cystein deficiency caused a reduction in C-oxygenation in strain R microsomes, and this reduction was nearly doubled by methyl mercury pretreatment of the depleted rats. Methyl mercury pretreatment per se of strain R rats on the standard diet gave no effect. By contrast microsomes from cystein deficient SpD rats showed no statistically significant decrease in C-oxygenation, and cystein deficiency did not further enhance the inhibitory effect obtained with methyl mercury pretreatment alone. N-oxygenation was not significantly affected by any treatment of the two strains.

Influence of dietary selenium on the mutagenic activity of perfusate and bile from rat liver, perfused with 1,1-dimethylhydrazine

The mutagenic effect of 1,1-dimethylhydrazine (UDMH) was studied in the liver perfusion/cell culture system. Male Wistar rats, fed a selenium-deficient diet with or without selenium supplementation in the drinking water, were used as liver donors. UDMH caused an increased mutation frequency in Chinese hamster V79 cells exposed in the perfusate. The effect was statistically significant with both selenium-deficient and selenium-supplemented livers. With selenium-deficient livers, a significant mutagenic effect was also obtained when V79 cells were treated with bile collected after the administration of UDMH. Bile flow and bile acid excretion were not affected by UDMH treatment of selenium-deficient or selenium-supplemented livers. There was a tendency towards reduced C-oxygenation of N,N-dimethylaniline in microsomes from selenium-deficient livers perfused with UDMH. The lactate/pyruvate ratio in the perfusate was increased by UDMH, the effect being more pronounced with selenium-deficient than selenium-supplemented livers.

Effects of dimethylnitrosamine on metabolism of N,N-dimethylaniline by rat liver microsomes: a comparative study of treatment in vivo, in isolated liver perfusion and in the microsomal system

The effect of dimethylnitrosamine (DMN) on rat liver microsomal detoxication was studied, using the non-carcinogenic aromatic amine N,N-dimethylaniline (dimethylaniline) as substrate. Prior to the preparation of microsomes, the rat liver was exposed to DMN either in vivo (by i.p. injection) or in the isolated liver perfusion system (by addition to the perfusion medium). DMN treatment in vivo (20 mg/kg body wt.) caused a 40% increase in dimethylaniline N-oxygenation and a 30% decrease in dimethylaniline C-oxygenation. When DMN was added to the perfusion medium to a final concentration of 5 or 25 mM, a similar effect was observed. With the 5 mM dose, C-oxygenation was decreased by 20% with a non-significant increase in N-oxygenation. The higher dose caused a 50% increase in N-oxygenation, whereas the decrease in C-oxygenation remained at 20%. When microsomes were incubated with both DMN (5 mM) and dimethylaniline (5 mM) in the system, a small but significant decrease in both N- and C-oxygenation of dimethylaniline was observed. The effect of DMN on the amino acid incorporation into liver and plasma proteins was also studied in the liver perfusion system. The synthesis of both liver and plasma proteins was reduced by DMN.