Comparison of co-cultivation of V79 cells with rat hepatocytes and rat H4IIE hepatoma cells for studying nitrosamine-induced hprt gene mutations.

The induction of mutations by nitrosamines in the hprt locus of V79 Chinese hamster cells was examined after metabolic activation in a co-cultivation system using either freshly isolated rat hepatocytes or H4IIE rat hepatoma cells and the results obtained were compared with systems which employ the rat liver microsomal fraction (S9-mix). This study was also designed as a first approach to investigating the induction of point mutations by tobacco-specific nitrosamines in mammalian cells in order to obtain information about the significance of these compounds in connection with the carcinogenicity of tobacco smoke. The mutagenicity of two tobacco-specific nitrosamines, 4-(methylnitroso)-1-(3-pyridol)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), were investigated and compared to two extensively investigated nitrosamines, i.e. dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). DMN was activated to mutagenic species by primary hepatocytes at mumolar concentrations, i.e. 1/100 of the concentrations required for mutagenesis by DEN and NNK. NNN was not activated to mutagenic species by liver S9 or primary hepatocytes. The findings shown here on the mutagenicities of NNK and NNN with liver preparations are in agreement with their relative carcinogenic potencies. When the established liver cell line H4IIE was used for metabolic activation, DMN and was found to be mutagenic, whereas the results for NNN were borderline and for DEN and NNK were without effect. The fate of these compounds via different metabolic pathways is discussed in terms of systems for detection of mutagenic metabolites and type of mutation induced.

Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin-permeabilization of hepatocytes isolated from control and clofibrate-treated mice.

Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble ('cytosolic') epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5-10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4-fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5-fold by clofibrate treatment and 15-35% of this activity is shifted from the peroxisomes to the cytosol.

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.

Correlation between induction of unscheduled DNA synthesis in the liver and excretion of mutagenic metabolites in the urine of rats exposed to the carcinogenic air pollutant 2-nitrofluorene.

The genotoxic effects of 2-nitrofluorene (NF) have been studied in vivo by measuring the induction of DNA repair, i.e. unscheduled DNA synthesis (UDS), in hepatocytes from male rats pretreated by oral gavage with NF. During the NF exposure, urine was collected for 24 h, and its mutagenicity was investigated in the plate incorporation assay, using Salmonella TA98 as tester strain. The urine samples were also used for the identification of excreted metabolites of NF. Rats treated with 2-acetylaminofluorene (AAF) were studied simultaneously. A positive UDS response was observed 12 and 24 h following a single gavage exposure to 12.5, 25 and 50 mg/kg NF, with the response returning to near control levels by 36 h. The positive control AAF induced approximately twice the response observed with NF, and both compounds gave a UDS response that was 2-3 times higher in Wistar rats relative to Sprague-Dawley rats. A potent direct-acting mutagenic effect was observed in urine samples after NF treatment, while AAF exposure only gave rise to a weak mutagenic effect, the NF/AAF ratio being 10/1. The stronger urinary mutagenicity after NF treatment relative to AAF treatment was associated with the presence of hydroxylated NFs. The genotoxic effect observed in the liver after NF treatment is, on the other hand, more likely due to the same AAF metabolites that are also formed after in vivo treatment with AAF.

Effects on in vivo and in vitro administration of vinblastine on the perfused rat liver--identification of crinosomes.

Livers of nonstarved rats were perfused for up to 4 hr in a recirculating system. Bile production, transaminases, and the lactate/pyruvate ratio remained at normal values. The ultrastructure of the hepatocytes was also well preserved even after the 4-hr perfusion. When vinblastine was given either in vivo or in vitro by addition to the perfusion fluid, it caused a conspicuous expansion of the autophagic-lysosomal compartment. Initially, nascent autophagic vacuoles developed, followed by the appearance of more mature ones and finally an increase in dense bodies was observed. In addition, administration of vinblastine in vivo gave rise to an increased occurrence of a subpopulation of lysosomes laden with VLDL-like particles. The term crinosomes seems appropriate for these lysosomal vesicles, since they apparently evolve by means of fusion between retained secretory granules and preexisting lysosomes (dense bodies). Addition of vinblastine of the perfusion fluid decreased the rate of proteolysis whether four times the serum concentration of amino acids were added or not. However, when vinblastine was given in vivo, proteolysis as measured in the perfusate decreased during the initial 3 hr of VBL treatment, whereas by longer times of pretreatment protein degradation exceeded the control value, constituting an example of catch-up proteolysis. Autophagic vacuoles isolated after short exposure to vinblastine in vivo exhibited high rates of protein degradation when incubated at acid pH. Insufficient proton pumping rather than lack of hydrolytic enzymes seems to be the most plausible explanation for this prompt pH effect.

Metabolism of the carcinogenic air pollutant 2-nitrofluorene in the isolated perfused rat lung and liver.

The metabolism of 2-nitrofluorene (NF), a model substance for nitrated polycyclic aromatic hydrocarbons, was studied in the isolated perfused rat lung and liver. NF has been identified in urban air and diesel exhaust and occurs in the gas, as well as in the particulate phase. Therefore, it is conceivable that the lung represents one point of entry of this compound into the body. The lung metabolizes NF to hydroxylated NFs, mainly 9-hydroxy-NF, independently of the route of administration (intravascular or intratracheal). After intratracheal administration, NF is rapidly excreted into the perfusate, indicating that other organs might be exposed to unmetabolized NF. The liver excretes NF metabolites as biliary glucuronides. Untreated bile is not mutagenic. However, after beta-glucuronidase treatment of bile, direct-acting mutagens were detected. The mutagenic metabolites in beta-glucuronidase-treated bile were the same as identified in the perfusate of the isolated lung. Since beta-glucuronidase is an enzyme found in the human intestinal microflora, inhalation of NF could result in the liberation of genotoxic metabolites in the colon.

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.

Effect of dietary selenium on biotransformation and excretion of mutagenic metabolites of N-nitrosodimethylamine and 1,1-dimethylhydrazine in the liver perfusion/cell culture system.

The mutagenicity of N-nitrosodimethylamine (NDMA) and 1,1-dimethylhydrazine (UDMH) has been studied in an isolated liver perfusion/cell culture system. The liver donors, male Wistar rats, were either selenium (Se)-deficient or had a physiologically adequate Se status (Se-supplemented). Mutagenicity was measured in perfusate and bile with Chinese hamster V79 cells as the genetic target. Se deficiency increased the mutagenic effect of NDMA in the perfusate, whereas no mutagenicity was detected in the bile of either Se-deficient or Se-supplemented livers. No significant increase in the mutagenicity of UDMH was seen in the perfusate with Se deficiency, but the bile became mutagenic. Se deficiency thus increased the mutagenicity of both NDMA and UDMH: with NDMA, the effect was observed in the perfusate, and with UDMH, in the bile.

Potent mitogenic activity of 4-acetylaminofluorene to the rat liver.

Administration of 4-acetylaminofluorene (4AAF) to rats by oral gavage (1000 mg/kg) produces a wave of S-phase activity in the liver 36 h later, followed by a wave of mitoses at 48 h. These events were monitored by autoradiography of isolated hepatocytes and by histopathology, respectively. DNA-labelling was shown to occur following both in vivo and in vitro radiolabelling. The level of S-phases observed approached that reported following partial hepatectomy. These effects were not accompanied by unscheduled DNA synthesis (UDS) nor was any frank histopathological damage to the liver evident. 2-Acetylaminofluorene (2AAF) elicited a very weak S-phase response at a dose level of 50 mg/kg, but gave marked UDS between 12 and 48 h.

Concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene.

Oral dosing of between 5-30 mg/kg of cyclophosphamide (CP) to Alderley Park rats induced micronuclei in the bone marrow between 12 and 36 h after dosing, but failed to induce unscheduled DNA synthesis (UDS) in the liver at similar dose levels and treatment periods. Dose levels of greater than 30 mg/kg were toxic to the liver. In contrast, 2-acetylaminofluorene (2AAF) induced UDS in the rat liver between 4-36 h after dosing, but gave only a weak response in the bone marrow assay at dose levels between 0.5 and 2 g/kg. Selected observations were made for each chemical using both tissues of the same test animal. It is concluded that an assessment of the genotoxicity in vivo of chemicals defined as genotoxic in vitro will contribute to an assessment of their possible mammalian carcinogenicity, and that these should involve assays conducted using both the bone marrow and the liver of rodents. Due to its relative ease of commission, the bone marrow micronucleus assay will usually be conducted first; in the case of negative results it is recommended that a liver genotoxicity assay should be conducted. The case for employing in vivo short-term genotoxicity tests to predict the possible organotropic carcinogenicity or germ cell mutagenicity of a new in vitro genotoxin is discussed.

Use of an in vivo/in vitro rat liver DNA repair assay to predict the relative rodent hepatocarcinogenic potency of 3 new azo mutagens.

The in vivo/in vitro rat liver DNA repair assay described by Mirsalis and Butterworth has been employed to compare the relative genotoxicity to the rat liver of three mutagenic analogues of the potent rat hepatocarcinogen 6-dimethylamino-phenylazobenzthiazole (6BT). The compounds evaluated were 6BT, its monomethyl analogue (6-monomethylamino-phenylazobenzthiazole; MA6BT), an analogue in which the -NMe2 group of 6BT is replaced by a piperidinyl group (6-[4-N-piperidinylphenyl]azobenzene; 6PT) and the N-cyanoethyl analogue of MA6BT (6-[p-(N-beta-cyanoethyl-N-methylamino)-phenylazo]benzthiazole; CNEt6BT). The order of relative carcinogenic potency predicted by the Salmonella mutation data was CNEt6BT much greater than MA6BT = 6BT much greater than 6PT. In contrast, that inferred from the in vivo liver DNA repair data was MA6BT greater than 6BT much greater than CNEt6BT much greater than 6PT. This divergence of predictions is discussed in terms of the differing solution properties of the four test chemicals.

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.

Dietary selenium deficiency causes decreased N-oxygenation of N,N-dimethylaniline and increased mutagenicity of dimethylnitrosamine in the isolated rat liver/cell culture system.

Male Wistar rats were fed diets of varying selenium content in order to obtain selenium-deficient and selenium-supplemented rats. After 5-6 weeks on the respective diet, the rats were used to investigate how selenium influences the effect of dimethylnitrosamine (DMN) on some liver enzymes and related reactions. The selenium-dependent glutathione peroxidase activity in postmicrosomal supernatant from liver was about 1% in selenium-deficient rats as compared to selenium-supplemented rats or rats fed a standard diet. The highest DMN-demethylase activity was observed in postmitochondrial supernatant from selenium-deficient rat liver, and the lowest in selenium-supplemented rats. No dietary effect was observed on hepatic microsomal cytochrome P450 levels. C-Oxygenation of N,N-dimethylaniline (DMA) was not affected by the selenium level. On the other hand, selenium deficiency seemed to reduce N-oxygenation of DMA. The mutagenicity of DMN in Chinese hamster V79 cells after metabolic activation by the isolated perfused rat liver, was approximately doubled when selenium-deficient livers were used as compared to selenium-supplemented livers and livers from rats fed a standard diet. A negative correlation between DMA-N-oxygenation and mutagenicity from DMN was observed, whereas no correlation between DMA-C-oxygenation and mutagenicity from DMN was found.

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.

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 liver's 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.

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.

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 Hubermann's 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.

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.

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.