Carcinogenic activity of benzo[a]pyrene in a 2 year oral study in Wistar rats.

Because of the relatively high human oral exposure to polycyclic aromatic hydrocarbons (PAHs) compared to the inhalation exposure, the known carcinogenicity of this type of compounds and the limited data from oral studies available with polycyclic aromatic hydrocarbons, an oral carcinogenicity study was performed using benzo[a]pyrene (B[a]P) as a PAH representative. Wistar rats, 52 animals per sex and group were exposed daily (5 days a week) to 0, 3, 10 or 30 mg B[a]P/kg bw/day by gavage for 104 weeks and were subject to gross- and histopathology. The main tumours observed were hepatocellular carcinomas and forestomach tumours. Other tumours induced in this study were tumours of the auditory canal, skin and appendages, oral cavity, small intestine, kidney, and soft tissue sarcomas. For hepatocellular carcinomas and forestomach tumours, the BMDL10 were 3 and 1 mg/kg bw/day, respectively. The incidence of altered hepatic foci was increased in the 3mg/kg bw/day group. The increase in liver tumours is considered the most relevant effect for human risk assessment in terms of pathogenesis and sensitivity, and is proposed as the basis for human cancer risk assessment for oral PAH exposure.

In vivo cytokinesis blocked micronucleus assay with carbendazim in rat fibroblasts and comparison with in vitro assays.

A successful in vivo application of the cytokinesis blocked micronucleus assay for the detection of aneuploidy induced by carbendazim (CARB) was carried out in the granuloma pouch assay. This was performed in two ways: (i) in vivo exposure of the skin fibroblasts to cytochalasin B (cytB) and CARB, by simultaneous injection of both substances into the pouch; (ii) in vivo exposure to CARB followed by in vitro culturing of the fibroblasts in the presence of cytB. Only the first assay was successful. Injection of cytB (with or without the test compound) into the pouch resulted in the induction of binucleate cells in vivo, up to a maximum of 5% at 1 mg cytB/pouch. After injection of CARB (0-50 or 0-10 mg/pouch) and cytB (1 mg) into the pouch, aneuploidy was determined in the isolated binucleate fibroblasts by fluorescence in situ hybridization with a general centromeric probe and combinations of chromosome-specific probes (19p + 19q, 4q + Yq). With all probes, the induction of chromosome loss and/or non-disjunction by CARB was very pronounced; at 10 mg CARB/pouch the total malsegregation frequency of chromosomes 4, 19 and Y was approximately 300/1000 binucleate cells. In an in vitro cytokinesis block assay with CARB (0-2.5 microg/ml) in primary skin fibroblasts the induced aneuploidy frequencies were as high as observed in the in vivo assay. The use of two probes for chromosome 19, which enabled the scoring of chromosome breaks in addition to aneuploidy, revealed no significant induction of chromosome breaks by CARB. The frequency of polyploid mononucleate and binucleate cells was decreased after CARB treatment, in both the in vivo and in vitro assays. However, in an additional in vitro assay without cytB a major induction of polyploidy from 2.5 microg/ml CARB and above was observed, showing that cytB may interfere with polyploidy induction.

Isolation of DNA probes specific for rat chromosomal regions 19p, 19q and 4q and their application for the analysis of diethylstilbestrol-induced aneuploidy in binucleated rat fibroblasts.

DNA probes specific for rat chromosomes 19p, 19q and 4q were isolated, characterized and used for the detection and analysis of diethylstilbestrol(DES)-induced aneuploidy. By denaturing and partially reassociating total genomic DNA a new rat repetitive DNA family was isolated, which was located on chromosome 19p21. Sequencing of a number of subclones from cos76-1 and other clones of this so-called 76-family revealed that the repeat units are interrupted with large areas of other (unique) DNA. Consequently, after fluorescence in situ hybridization (FISH) the signals in interphase nuclei are large and spread out. The other two probes, cos25 (chromosome 4q) and cos42-47 (chromosome 19q), were isolated by screening cosmid libraries with probes isolated previously in our laboratory. The repeat unit of cos25 is a 2174 bp long EcoRI unit that contains three Sau3A sites and is tandemly organized. Sequencing of subclones of cos42-47 revealed that this probe was in fact the 5S RNA gene, located on 19q12. In order to determine if these probes were suitable probes for aneuploidy detection, two series of dual colour FISH with the combinations cos25/cos76-1 (4q/19p) and cos42-47/cos76-1 (19q/19p) were carried out on slides from an in vitro micronucleus assay with DES. With all three probes used, an increase in binucleated cells with non-disjunction or chromosome loss was observed in the DES-treated cultures. Scoring of additional micronucleated cells on slides hybridized with the cos25/cos76-1 (4q/19p) probes revealed that the hybridization signal of probe cos25 (4q) was over-represented in the micronuclei of the control cultures. The simultaneous use of the 19q and 19p probes is a particularly valuable tool for the detection of aneuploidy, since it allows distinction between aneugenic and clastogenic events in binucleated cells. Results of this analysis showed that apart from aneuploidy, DES also induced structural chromosome aberrations, although to a lesser extent.

Increased frequencies of diploid sperm detected by multicolour FISH after treatment of rats with carbendazim without micronucleus induction in peripheral blood erythrocytes.

The purpose of the present study was to determine the effect of a single oral dose of carbendazim (CARB) on the frequencies of numerical chromosome aberrations in sperm and on micronuclei in peripheral blood erythrocytes of rats. Dual colour FISH on epididymal sperm of rats treated 31 days before sacrifice (0, 50, 150, 450 and 800 mg/kg body wt CARB in corn oil), corresponding to exposure during late pachytene, revealed a clear induction of diploid sperm. Induction of aneuploid sperm was not observed. Although the absolute frequencies of diploidy were low, ranging from 0.03% in the control group to 0.22% in the highest dose group, the observed dose-response relationship was highly significant. In sperm of rats killed 50 days after treatment with CARB (corresponding to exposure of spermatogonial stem cells) the effect was no longer apparent. In a second experiment, in addition to more dose groups in the low dose range, the peripheral blood micronucleus assay was incorporated. Results of triple colour FISH on epididymal sperm of rats treated with CARB (0-800 mg/kg body wt) again showed induction of diploid, but not of aneuploid sperm. Induction was less prominent than in the first experiment, but the dose-response relationship for diploidy was again significant. In blood samples drawn from the tail vein 48 h after treatment with CARB induction of micronuclei in peripheral blood erythrocytes was not observed, whereas the micronucleus frequency was significantly increased after a single i. p. dose of mitomycin C (3 mg/kg body wt). In conclusion, the present results show that CARB induces diploidy in sperm, without an accompanying induction of micronuclei in erythrocytes. This finding suggests that in rats the peripheral blood micronucleus assay is a less sensitive indicator for the genotoxic potential of CARB than the epididymal sperm aneuploidy/diploidy assay.

Analysis of DES-induced micronuclei in binucleated rat fibroblasts: comparison between FISH with a rat satellite I probe and immunocytochemical staining with CREST serum.

The usefulness of fluorescence in situ hybridization (FISH) with rat satellite I DNA was compared with immunocytochemical staining with CREST serum for the analysis of the content of micronuclei from primary rat fibroblasts. We analyzed micronuclei induced in vitro by the aneugenic compound diethylstilbestrol (DES) or the clastogenic compound mitomycin C (MMC). Since a centromeric probe was not available for the rat, we isolated rat satellite I DNA by PCR with primers designed on the basis of the known rat satellite I DNA sequence. The PCR products obtained as well as the cloned PCR products showed hybridization to the centromeric regions of a large number of chromosomes, but not of chromosome 1, 19, 20, X and Y. Clone 18-5 was further analyzed and was shown to contain at least 4 repeats of the rat satellite I family. This probe, which hybridizes in the centromeric region of 34 of the 42 chromosomes, was used throughout the study as a probe for the FISH analysis of the micronuclei. For the immunocytochemical staining, the commonly used commercial anti-centromeric antibodies could not be used because of the weakness of the fluorescent signals given. Consequently, CREST serum of a single patient was used, which showed bright and distinct signals on the kinetochores of each chromosome. After treatment of the cells with the aneugen DES an increase in centromere (FISH) and kinetochore (CREST) positive micronuclei was found, whereas after treatment with the clastogen MMC, the percentage of centromere-positive micronuclei was similar to that observed in controls. Analysis of a large number of DES-induced micronuclei showed that the immunocytochemical method is equally as or slightly less sensitive for the detection of chromosomes in micronuclei and we therefore recommend FISH with probe 18-5 for the detection of chromosome loss in rat cells.

The detection and evaluation of aneugenic chemicals.

Although aneuploidy makes a significant contribution to both somatic and inherited disease the mechanisms by which environmental chemicals may induce numerical chromosome aberrations are only poorly defined. The European Union Project was aimed to further our understanding of those chemical interactions with the components of the mitotic and meiotic cell division cycle which may lead to aneuploidy and to characterise the parameters such as cellular metabolism which may influence the activity of aneugenic chemicals. C-mitosis can be induced by the highly lipophilic polychlorinated biphenyl and the completion of mitosis and cleavage can be modified by agents which deplete cellular levels of reduced glutathione. Modifications of the fidelity of chromosome segregation were produced by inhibiting the functioning of topoisomerase II during chromatid separation. In contrast, the modification of centromere integrity resulted in chromosome breakage as opposed to disturbance of segregation. Modifiers of tubulin assembly and centriolar functioning in somatic cells such as acrylamide, vinblastine and diazepam reproduced their activity in rodent bone marrow and male germ cells. The analysis of chromosome malsegregation in Aspergillus nidulans by a structurally related series of halogenated hydrocarbons was used to develop a QSAR model which had high predictive value for the results of fungal tests for previously untested related chemicals. Metabolic studies of potential aneugens in genetically engineered human lymphoblastoid cells demonstrated the detoxification of the aneugenic activity of chloral hydrate and the activation of 2,3-dichlorobutane, 1,1,2-trichloroethane and trichloroethylene by Phase I biotransforming enzymes. Cell transformation studies in Syrian hamster dermal cultures using a panel of 22 reference and or potential aneugens indicated that 15 of the 22 produced positive results following single exposures. Five of the aneugens which were negative following single exposures produced positive results where cultures were continuously exposed for up to 6 weeks to low concentrations following a single non-transforming exposure to the mutagen dimethyl sulphate. The transformation studies indicate that a significant proportion of chemical aneugens are potential complete carcinogens and/or co-carcinogens. To optimise the enumeration of chromosomes following exposure to potential chemical aneugens whole chromosome paints and centromere specific probes suitable for use in fluorescence in situ hybridisation (FISH) were developed for the rat, mouse and Chinese hamster and selected human probes evaluated for their suitability for routine use. Molecular chromosome probes were used to develop protocols for enumerating chromosomes in metaphase cells and centromeres and micronuclei in interphase cells. The analysis of segregation of specific centromeres in binucleate cells following cytochalasin B treatment was shown to be a potentially valuable system for characterising non-disjunction following chemical exposure. Whole chromosome paints and centromere specific probes were used to demonstrate the presence of dose-response thresholds following treatment with a reference panel of spindle inhibiting chemicals. These data indicate that the FISH technology is suitable for evaluating the relative hazards of low-dose exposures to aneugenic chemicals.

Gamma-radiation-induced mutation spectrum in the episomal lacI gene of Escherichia coli under oxic conditions.

In this study we have determined the mutation spectrum in the complete episomal lacI gene of Escherichia coli induced by gamma-radiation under oxic conditions. Mutants were generated by 60Co gamma-irradiation of an E. coli culture of stationary cells in LB medium, under continuous flushing with oxygen. Oligonucleotide probe analysis showed that 14% of the gamma-ray-induced mutations were located at the lacI gene hot spot at position 620-632, which is characterized by a triple repeat of the 5'-TGGC-3' sequence. Previously it was shown that about 70% of the spontaneous mutations were located at this site due to the loss or the addition of a TGGC sequence. The non-hot spot mutations were further characterized by automated sequence analysis. The results show that base pair (bp) substitutions were the main type of gamma-ray-induced mutations. Although all types of bp substitutions were observed, 74% of the bp substitutions involved C/G base pairs. C/G --> T/A and C/G --> A/T substitutions were predominant, both accounting for 35% of all bp substitutions, whereas A/T --> C/G substitutions were only seldomly observed (3%). A relatively large amount of -1 bp deletions (15% of all mutations) was detected in the gamma-ray-induced mutation spectrum, mainly affecting C/G base pairs, and 10% were deletions, ranging in size from 11 to 532 bp. It can be concluded that under oxic conditions gamma-radiation induces in E. coli mainly bp substitutions of all types but preferentially at C/G base pairs, and that the mutations tend to be randomly distributed within the lacI gene sequence.

Detection of point mutations in T lymphocytes.

We exposed experimental animals to a series of alkylating agents that induced mutations at the X-linked hprt gene of T lymphocytes. We then isolated the mutant cells and analyzed the molecular nature of the mutations by amplification of hprt cDNA sequences with the use of reverse transcriptase PCR followed by DNA sequence analysis, and then correlated the mutational spectra obtained to the spectra of DNA adducts caused by the alkylating agents used. The nature of the base-pair changes causing the mutations was characteristic for the reaction pattern of the genotoxic agent with DNA. However, we also found a clear influence of DNA repair processes; i.e., in those cells that were able to remove certain types of DNA damage, the class of mutations expected from that type of damage was reduced.

Marked differences in the role of O6-alkylguanine in hprt mutagenesis in T-lymphocytes of rats exposed in vivo to ethylmethanesulfonate, N-(2-hydroxyethyl)-N-nitrosourea, or N-ethyl-N-nitrosourea.

The role of DNA alkylation at the O6 position of guanine in the induction of gene mutations in vivo was studied in the hprt gene of rat T-lymphocytes from spleen exposed in vivo to the monofunctional ethylating agents ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU), or the hydroxyethylating agent N-(2-hydroxyethyl)-N-nitrosourea (HOENU). All chemicals showed an exposure-dependent increase in hprt mutant frequency. HOENU and ENU, however, were much more mutagenic than EMS when compared at equimolar levels. DNA sequence analysis was performed on PCR products of hprt cDNA from 40 EMS-, 35 HOENU-, and 46 ENU-induced 6-thioguanine-resistant T-lymphocyte clones. Thirty EMS-induced mutants contained a single base pair substitution with GC to AT transitions being the predominant type of mutation (26 of 30) which are probably caused by mispairing of O6-ethylguanine with T during DNA replication. No strand specificity of mutated G's among GC to AT transitions was observed. Twenty-three HOENU- and 42 ENU-induced mutants contained a single base pair substitution. In contrast to EMS, GC to AT transitions were found at a low frequency, 4 of 23 for HOENU and 5 of 42 for ENU, indicating that O6-hydroxyethylguanine and O6-ethylguanine are less important in HOENU- and ENU-induced mutagenesis in vivo, respectively. Also here no strand bias for mutated G's was observed, although the number of this type of mutation was limited. The most frequently induced base pair alterations by HOENU and ENU were transversions at AT base pairs, 16 of 23 and 28 of 42, respectively, with AT to TA being the predominant type of mutation. In both ENU and HOENU mutational spectra, an extreme strand bias for mutated T's toward the nontranscribed strand was found. The results suggest that DNA damage induced in rat T-lymphocytes in vivo by HOENU and ENU is processed in similar ways.

AT base pairs are the main target for mutations at the hprt locus of rat skin fibroblasts exposed in vitro to the monofunctional alkylating agent N-ethyl-N-nitrosourea.

Spectra of N-ethyl-N-nitrosourea (ENU)-induced mutations differ widely among various in vitro and in vivo mutational systems. To investigate possible reasons for these differences, a mutational system is needed in which the same target gene is used for comparison in the same type of cells in vitro and in vivo. In the present study, this was achieved by analysing at the molecular level 35 hprt mutant rat fibroblast clones obtained from cell populations exposed in vitro to ENU and comparing the mutational spectrum with the previously determined spectrum of ENU-induced hprt mutants in the same target cells exposed in vivo. Twenty-eight mutants contained a single base pair alteration in the hprt coding sequence. Most of these changes were found at AT base pairs (19/28), the AT to TA transversion being the most frequent kind of mutation (12/19), which is probably caused by O2-ethylthymine. Transversions at AT base pairs showed all mutated T's to be located in the nontranscribed strand of the hprt gene, suggesting a strand specific fixation of mutations induced by O2-ethylthymine, which appears to be a general feature of ENU- and ENNG-induced hprt mutations in mammalian cells. GC to AT transitions, probably caused by O6-ethylguanine, were detected at a lower frequency (7/28). This in vitro mutational spectrum was very similar to that of the same target cells exposed in vivo to ENU. A comparison of the mutational spectra in AGT-proficient and AGT-deficient rodent cells exposed to ethylating agents showed that in contrast to the situation in AGT-proficient rat fibroblasts, GC to AT base pair changes (and not AT to TA) are the predominant mutations in AGT-deficient hamster cells.

Formation and persistence of DNA adducts in pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

Base substitutions and frameshifts induced by genotoxic agents are considered to result mainly from incomplete repair and incorrect replication of modified nucleotides in DNA. In this study, induction and persistence of O6-alkyl- and 7-alkylguanine adducts were determined by reverse phase HPLC and electrochemical detection in DNA of pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU). Although an exposure dependent increase in the level of adducts was found for both chemicals, a much lower frequency of both O6-alkylguanine and 7-alkylguanine was detected after ENU treatment than after MNU treatment, indicating that MNU is much more reactive with DNA than ENU. The persistence of O6-alkyl- and 7-alkylguanine was studied for up to 48 h at exposure levels of 60 mg/kg for MNU and 100 mg/kg for ENU. A time-dependent decline in the levels of both adducts was observed, but w6-alkylguanine was more rapidly lost than 7-alkylguanine in both pouch skin fibroblasts and liver. Furthermore, DNA adducts were faster lost from liver than from pouch skin fibroblasts. The loss of O6-alkylguanine adducts is probably mediated by the action of O6-alkylguanine-DNA alkyltransferase (AGT) in the target tissues since AGT activity was detectable in protein extracts of pouch skin fibroblasts and liver from unexposed rats and from exposed rats, 48 h but not 1 h after MNU and ENU treatment. AGT activity recovered faster in liver tissue than in pouch skin fibroblasts, and after ENU exposure an induction of AGT activity was observed in the liver but not in pouch skin fibroblasts. The difference in the level of O6-alkylguanine in DNA of pouch skin fibroblasts introduced upon exposure to MNU and ENU may explain the molecular nature of most base pair changes observed previously in spectra of hprt mutants induced in these cells in vivo. The frequency of O6-methylguanine upon MNU exposure remains relatively high with time and these adducts most likely cause GC to AT transitions. In the case of ENU, O6-ethylguanine was detected at very low frequencies resulting in a low contribution of GC to AT transitions. Rather, the ENU spectrum is dominated by base pair changes at AT base pairs.

Molecular analysis of hprt gene mutations in skin fibroblasts of rats exposed in vivo to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

The granuloma pouch assay in the rat is a model system in which relative frequencies of genetic and (pre-) neoplastic changes induced in vivo by carcinogenic agents can be determined within the same target tissue. The target is granuloma pouch tissue and consists of a population of (transient) proliferating fibroblasts which can be cultured in vitro. hprt gene mutations were studied in granuloma pouch tissue of rats treated with single doses of direct acting alkylating agents N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an exposure-dependent increase in the hprt mutant frequency. Thirty-seven MNU (60 mg/kg)- and 43 ENU (100 mg/kg)-induced hprt mutant cell clones were analyzed at the molecular level. Twenty-two MNU-induced and 36 ENU-induced mutants carried a single base pair change in exon sequences of the hprt gene. The predominant base pair alterations induced by MNU were GC to AT transitions (18 of 22), which are probably caused by O6-methylguanine lesions. For most of the GC to AT transitions (16 of 18), the G was located in the nontranscribed strand, suggesting a strand bias in the repair of O6-methylguanine lesions. ENU-induced mutations occurred predominantly at AT base pairs (28 of 36), being mostly AT to TA and AT to CG transversions, and are probably caused by O2-ethylthymidine. Also here, DNA repair processes seem to act with different rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since all the 24 transversions observed at AT base pairs had the thymidine residue in the nontranscribed strand. GC to AT transitions were only present at a low frequency among ENU-induced mutations, suggesting that O6-ethylguanine lesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU- and ENU-induced hprt mutant clones were different from spontaneously occurring hprt mutant clones. These results indicate that MNU and ENU induce different mutational spectra in vivo and that DNA repair systems remove O6-methylguanine, O2, and/or O4-ethylthymidine much faster from the transcribed strand than the nontranscribed strand of the hprt gene in these rat fibroblasts.

Chromosomal localization of the rat N-ras protooncogene by fluorescence in situ hybridization.

The rat N-ras protooncogene has been assigned to chromosome 2q34 by fluorescence in situ hybridization on rat metaphase chromosomes. This was accomplished using two recently isolated genomic clones with a length of 8.2 and 4.1 kb.

The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.

Role of genotoxicity assays in the regulation of chemicals in The Netherlands: considerations and experiences.

This paper discusses genotoxicity testing and data interpretation as applied in The Netherlands in the context of the regulation of chemicals. Guidelines were first formulated in 1981 and their use evolved in practice, on the basis of increasing experience at the national and international levels. The distinction between in vitro assays to detect intrinsic genotoxic properties and in vivo assays as a subsequent phase to show the realization of this potential in an intact organism has always been a cornerstone of the Dutch approach. Several critical aspects of the use of short-term genotoxicity tests in sequential schemes are discussed, such as their predictivity for carcinogenicity, the limited database concerning the performance of short-term in vivo assays, the relevance of devising separate strategies to test for possible carcinogenicity and germ cell mutagenicity, and the use of short-term tests to discriminate between genotoxic and non-genotoxic carcinogens. Examples are given of how short-term tests contributed to the toxicological evaluation of chemicals in The Netherlands.

Evaluation of the DNA-repair host-mediated assay. III. Relationship between metabolic activation of dimethylnitrosamine and organ-specific differential lethality induced in E. coli indicator strains.

In the present study the sensitivity of differential lethality as an endpoint for monitoring the presence of organ-specific genotoxic factors within the DNA-repair host-mediated assay (HMA) was determined. The induction of differential lethality in chemically exposed animals was assessed by measuring the recovery ratio Q, i.e., the relative survival of a repair-deficient E. coli K-12 derivative in comparison with its repair-proficient counterpart. Using untreated animals the interindividual fluctuation of the recovery ratio Q was first quantified and then used to determine the level below which it could be considered indicative of chemically induced differential lethality. This Q value was found to be 0.65 or lower. Using this criterion, a significant decrease of the Q value was observed in mice exposed to DMNA at a dose level as low as 15-30 mumole/kg, i.p. Inter-organ transport (liver----extrahepatic organs) of indicator bacteria was studied in reconstruction experiments using the direct-acting methylating agent MNU. These studies showed that inter-organ transport of indicator bacteria did not interfere with MNU-induced differential lethality. Time-related experiments were used to study the effects of inter-organ transport of genotoxic DMNA metabolites. In these studies significant, time-related differences were found in the induction of differential lethality in various organs of mice treated with DMNA. At a dose level of 200 mumole/kg (i.p.) genotoxic factors appeared within 25 min after administration in the liver. In the lungs and kidneys such factors appeared at a substantially slower rate, e.g., 20-120 min after DMNA administration. In persistence experiments differential lethality reached a maximum 30 min after DMNA treatment. No residual effects were detected 60 min after the injection of the carcinogen. These experiments showed that DMNA-derived genotoxic factors diffused from the liver into the bloodstream. The diffusion of these reactive species followed by their transport via the bloodstream to the lungs accounted for maximally 50% of differential lethality observed in bacteria recovered from the latter organ. In contrast, no indications were found for the transport of genotoxic DMNA metabolites from the liver via the bloodstream to the spleen and the kidneys. These results show that organ-specific effects observed in the DNA-repair HMA procedure after DMNA exposure can be primarily attributed to in situ metabolism, rather than diffusion of genotoxic metabolites from the liver to extrahepatic organs.

Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli H. V: DNA sequence analysis of mutations in bacteria recovered from the liver of Swiss mice exposed to 1,2-dimethylhydrazine, azoxymethane, and methylazoxymethanolacetate.

The host-mediated assay (HMA) was used to determine the spectra of mutations induced in the lacl gene of Escherichia coli cells recovered from the livers of Swiss mice exposed to the carcinogens 1,2-dimethylhydrazine (SDMH), azoxymethane (AOM), and methylazoxymethanolacetate (MAMA). These spectra were further compared with changes induced by dimethylnitrosamine (DMNA) in the HMA methodology. A total of 177 independent lacl mutations arising in the HMA following exposure to SDMH, AOM, and MAMA were analyzed. Single-base substitutions accounted for 97% of all mutations analyzed. The vast majority of the single-base substitutions consisted of G:C----A:T transitions (94% of all mutations). The remaining mutations consisted of A:T----G:C transitions (3% of all mutations) while non-base substitutions accounted for only 3% of the total mutagenesis. The latter mutations consisted of one frameshift mutation and four lacO deletions. The distribution of G:C----A:T transitions induced by the three chemicals in the first 200 bp of the lacl gene was not random, but rather clustered at sites where a target guanine was flanked at the 5' site by a purine residue.