In vivo genotoxicity and acute hepatotoxicity of 1,2-dichloroethane in mice: comparison of oral, intraperitoneal, and inhalation routes of exposure.

The in vivo genotoxicity of 1,2-dichloroethane (DCE) was studied in the liver of male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice after single p.o., i.p., and inhalation exposures. The acute hepatotoxicity of DCE was also examined in order to determine nonnecrogenic exposure levels for each route of administration. Single-strand breaks and/or alkali-labile lesions were demonstrated by alkaline DNA-unwinding/hydroxylapatite chromatography in hepatic DNA at 4 hr after p.o. or i.p. administration of nonnecrogenic doses (100 mg/kg, p.o.; 150 mg/kg, i.p.) of DCE to groups of four to six mice. No evidence of hepatic DNA damage was found immediately following 4-hr inhalation exposures of mice to a nonnecrogenic (150 ppm) or necrogenic (500 ppm) concentration of DCE. Four-hr inhalation exposures of mice to concentrations of DCE causing high mortality within 24 hr (1000 to 2000 ppm) produced evidence of hepatic DNA damage at 4 hr, but the possibility that this damage was due to the acute necrogenic effects of the exposures could not be excluded. A significant fraction of the hepatic DNA damage observed 4 hr after i.p. administration of DCE (200 mg/kg) was still evident after 24 hr, indicating the persistence of unrepaired lesions in the DNA. These findings are consistent with the seemingly contradictory results of the two long-term carcinogenicity bioassays, in which DCE was found to be carcinogenic to Osborne-Mendel rats and B6C3F1, mice when administered by gavage but nontumorigenic to Sprague-Dawley rats and Swiss mice after chronic inhalation exposure. Therefore, our results provide additional evidence for the importance of a route of administration effect in the in vivo genotoxicity and carcinogenicity of DCE.

Malignant transformation of a preneoplastic hamster epidermal cell line by the EJ c-Ha-ras-oncogene.

We have investigated whether the activation of endogenous ras genes is associated with the immortalization or malignant transformation of primary hamster epidermal cells by chemical carcinogens. We have also asked whether transfection of a cloned c-Ha-ras oncogene (pEJ) into a nontumorigenic cell line established from hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine treatment can induce conversion to a malignant phenotype. DNA from the nontumorigenic epidermal cell line (H5-MNNG) and from two neoplastic cell lines transformed by benzo(a)pyrene was not capable of transforming NIH/3T3 cells. This result suggests that these cells do not contain an activated (mutated) ras gene. However, when H5-MNNG cells were cotransfected with pEJ and pSV2-gpt, a plasmid containing the dominant selectable marker gene Ecogpt, seven of nine clones of Ecogpt transformants formed carcinomas in nude mice and colonies in soft agar. Southern blot analysis of BamHl-digested genomic DNA from the Ecogpt-transformed clones indicated that rapid malignant transformation was associated with integration of a complete copy of the 6.6-kilobase fragment of pEJ containing the activated c-Ha-ras gene. Furthermore, DNA from the malignant clones transformed NIH/3T3 cells in a secondary transfection assay. These studies demonstrate that a mutated c-Ha-ras gene, under the transcriptional control of its normal cellular promoter, can rapidly transform a nontumorigenic epidermal cell line. This result suggests that activation of an endogenous c-ras gene can function as the final completing event in the progression of epithelial cells to the malignant phenotype. Thus, preneoplastic cell lines of both mesenchymal and epithelial origin have now been shown to be susceptible to malignant conversion by a single mutation in a c-Ha-ras proto-oncogene.

DNA fingerprinting of 7,12-dimethylbenz[a]anthracene-induced and spontaneous CD-1 mouse liver tumors.

Determining to what degree chemicals and environmental agents contribute to the development of cancer would be materially enhanced by the ability to distinguish chemically induced tumors from those that arise spontaneously. Using DNA fingerprinting as an assay, we investigated whether somatic DNA rearrangements are more frequent in chemically induced mouse liver tumors than they are in spontaneous mouse liver tumors. Tumors were induced by a single i.p. injection of 12-day old male Crl:CD-1(ICR)BR (CD-1) mice with 20 nmol/g 7,12-dimethylbenz[a]-anthracene and were harvested 9 to 12 months after injection. Spontaneous tumors were obtained from 94- to 98-week old male CD-1 mice. We detected 8 rearrangements in 14 7,12-dimethylbenz[a]anthracene-induced tumors, which corresponds to a high rearrangement frequency of about 2% (of the minisatellite bands examined). Furthermore, 6 of these rearrangements included complete band losses which must have occurred early in tumor development. However, only 2 band changes were observed in 15 spontaneous tumors, and both changes were intensity shifts which may represent rearrangements that occurred later during tumor progression. Histological examination showed that the higher frequency of rearrangements in 7,12-dimethylbenz[a]anthracene-induced tumors versus spontaneous tumors was not related to differences in the degree of tumor progression or malignancy. Our results suggest that DNA fingerprinting may be a valuable assay for differentiating certain chemically induced tumors from spontaneous tumors.

Activation of the Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice.

We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.

Rapid induction of an experimental metastatic phenotype in first passage rat embryo cells by cotransfection of EJ c-Ha-ras and c-myc oncogenes.

First passage rat embryo cells were transfected with plasmids carrying a mutated EJ c-Ha-ras oncogene alone or in combination with the c-myc oncogene. Three days later, unselected cultures were harvested and injected into nude mice either subcutaneously to assay for tumorigenicity or intravenously to assay for metastasis to the lung. The results indicate that a transcriptionally-enhanced EJ-c-Ha-ras oncogene alone can convert normal rat cells to a tumorigenic but not metastatic phenotype. Co-transfection of a c-myc oncogene with the EJ c-Ha-ras oncogene was necessary to produce the rapid, phenotypic conversion of normal cells to transformed cells with both tumorigenic and metastatic potential. No tumors were observed in animals injected with c-myc-transfected cells. Cell lines established from EJ c-Ha-ras-induced shoulder tumors were metastatic when reinjected intravenously into nude mice. These results support the hypothesis that the cooperative action of c-Ha-ras and c-myc oncogenes is more potent in inducing malignant transformation than either oncogene acting alone. Our results also suggest that the phenotypic conversion of normal cells to tumorigenic cells with experimental metastatic potential by ras and myc oncogenes can be completed within 3-4 cell divisions after transfection.

Rapid DNA degradation in primary rat hepatocytes treated with diverse cytotoxic chemicals: analysis by pulsed field gel electrophoresis and implications for alkaline elution assays.

The use of genetic toxicology tests for hazard identification is complicated by the fact that some in vitro tests using cultured mammalian cells are subject to potential artifacts that can make it difficult to distinguish between direct, chemically-induced genotoxicity, and DNA damage that occurs secondary to chemically-induced cytotoxicity (e.g., mediated by endogenous nucleases). Recently, we demonstrated that cytotoxicity-induced DNA double strand breaks (dsb) can produce artifacts in the in vitro alkaline elution/rat hepatocyte assay [Elia et al., 1993]. To explore this further, we used pulsed field gel/DNA dsb assays to characterize the relationship between chemically-induced cytotoxicity and the degradation of genomic DNA to high molecular weight fragments. Two sets of compounds were tested: 17 cytotoxic agents judged to be neither genotoxic nor carcinogenic, and 10 known genotoxic carcinogens. We found a close correlation between chemically-induced cytotoxicity and the rapid degradation of DNA to high molecular weight, double-stranded fragments. In contrast, the classic genotoxic chemicals tested generally did not trigger DNA dsb fragmentation at doses that were genotoxic but not immediately cytotoxic. These data indicate that pulsed field gel/DNA dsb assays can be used with in vitro genetic toxicology assays to help distinguish between genotoxic and cytotoxic mechanisms of DNA damage.

Current status and use of short/medium term models for carcinogenicity testing of pharmaceuticals--scientific perspective.

Short- and medium-term rodent bioassays have been proposed under ICH guidelines for use in testing for the carcinogenic potential of pharmaceuticals. Further evaluation of these models is needed urgently and coordinated efforts are in progress worldwide to expand the available database. Models currently being investigated include transgenic mice (Tg-rasH2, Tg.AC, p53(+/-), XPA(-/-)) and neonatal mice. As more data become available on the performance of these assays, regulatory and industry scientists will be faced with the difficult challenge of determining how the performance (accuracy) of each assay will be measured and deciding which assays have value in the risk assessment process.

Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan).

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5' nuclease PCR (TaqMan) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7kb beta-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using 32P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas 32P-postlabeling with nuclease P(1) enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B(1). Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing 32P-postlabeling assay with nuclease P(1) enrichment.

The effect of deuterium labeling on the genotoxicity of N-nitrosodimethylamine, epichlorohydrin and dimethyl sulfate.

Deuterated and non-deuterated N-nitrosodimethylamine, epichlorohydrin and dimethyl sulfate were evaluated for the ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. Non-deuterated nitrosodimethylamine induced twice the amount of DNA-strand breaks as the deuterated form. No evidence of a deuterium isotope effect was seen for the direct-acting alkylating agents epichlorohydrin and dimethyl sulfate.

Detection of DNA damage induced by human carcinogens in acellular assays: potential application for determining genotoxic mechanisms.

Positive outcomes of in vitro genotoxicity tests may not always occur as a consequence of direct reaction of a compound or a metabolite with DNA. To follow-up positive responses in in vitro tests, we developed two supplemental, cell-free assays to examine the potential of compounds and metabolites to directly damage DNA. Calf thymus DNA was used as the target for the direct detection of adducts by 32P-postlabeling/TLC and electrochemical detection, and alkaline gel electrophoresis was used to detect single-strand breakage of bacteriophage lambda DNA. To show that these assays would detect damage from relevant compounds, we examined nine human carcinogens (aflatoxin B1, busulfan, chlorambucil, cyclophosphamide, diethylstilbestrol, melphalan, 2-naphthylamine, phenacetin and potassium chromate). Each of the nine compounds produced a positive result for one or both endpoints. Using multifraction contact-transfer TLC, we detected 32P-labeled DNA adducts produced by aflatoxin B1, chlorambucil, diethylstilbestrol, melphalan, 2-naphthylamine, and potassium chromate (plus hydrogen peroxide). Aflatoxin B1, diethylstilbestrol and 2-naphthylamine required metabolic activation (induced rat liver S9) to generate DNA adducts. Although potassium chromate alone induced a slight increase in the content of 8-hydroxydeoxyguanosine (a promutagenic adduct produced by reactive oxygen species), addition of hydrogen peroxide greatly increased 8-hydroxydeoxyguanosine levels. The damage to lambda DNA by each human carcinogen (or metabolites), except diethylstilbestrol, was sufficient to generate single-strand breaks after neutral thermal hydrolysis at 70 degrees C. Chromate was a weak inducer of DNA fragmentation, but adding hydrogen peroxide to the reaction mixtures dramatically increased the DNA strand breakage. Our data suggest that these non-routine, acellular tests for determining direct DNA damage may provide valuable mechanistic insight for positive responses in cell-based genetic toxicology tests.

The mouse lymphoma L5178Y Tk+/- cell line is heterozygous for a codon 170 mutation in the p53 tumor suppressor gene.

The p53 tumor suppressor protein plays an important role in regulating the cellular response to DNA damage, including cell cycle arrest and apoptosis induction. Normal p53 function is critical for the maintenance of genomic stability. The mouse lymphoma L5178Y/TK(+/-)-3.7.2C cell line is widely used in genetic toxicology for mutagenesis and clastogenesis testing. A related line L5178Y-R, has previously been shown to react with antibodies specific for mutant as well as wild-type p53 protein and to exhibit delayed cell death after radiation. For this reason, as well as the mouse lymphoma assay's reputation for high sensitivity of detection for genotoxic agents but low specificity, we examined several clones of L5178Y cells for mutations in the conserved core domain (exons 5-8) of the p53 gene. Using single-strand conformational polymorphism analysis, we found evidence for the same mutation in exon 5 of p53 in L5178Y-R, L5178Y-S and L5178Y/TK(+/+)-3.7.2C cells. The mutation was identified by sequencing of exon 5 as a TGC (Cys) to CGC (Arg) transition in codon 170 (= codon 176 in humans). Sequencing showed approximately equivalent signals for the mutant and normal alleles for all 3 lines. The mutation in codon 170 is adjacent to a mutation hotspot of the human p53 gene (codon 175) and eliminates a critical zinc-coordinating cysteine residue such that the mutant protein is likely to be denatured and have a dominant negative effect on normal p53 function. Western blots showed approximately 100-fold higher levels of p53 protein in unirradiated L5178Y cells as compared to induced levels of p53 in normal mouse splenocytes 4 h after 5 Gy of gamma radiation. The high levels of p53 protein in L5178Y cells were not further inducible by radiation, whereas an 11-fold induction was seen in the irradiated splenocytes. These results indicate that p53 protein in L5178Y cells is dysfunctional and suggest that this line may therefore be abnormally susceptible to the induction of genetic alterations.

Co-purification of gastric mucoproteins with DNA: an explanation for the reported 'interaction' of omeprazole with DNA in rat tissues.

Recently, Phillips et al. reported that small amounts of radioactivity derived from [14C]omeprazole were 'associated' with DNA purified from gastrointestinal tissues of treated rats (Mutagenesis 7, 277-283, 1992). We hypothesized that this radioactivity arose from omeprazole bound to contaminating protein in the DNA fraction (Mutagenesis 7, 395-396, 1992). Using rats injected with 35S-labeled amino acids, we found significant protein contamination (0.06 microgram of protein per microgram of DNA) in DNA purified from gastrointestinal tissues. Gastric mucous proteins represent likely candidates for binding of omeprazole in the rat model used by Phillips et al. To investigate this, we partially purified proteins from gastric mucus, incubated them with [14C]omeprazole, and then added these radiolabeled mucoproteins to homogenates of rat colon and duodenum before starting the DNA purification. Detectable amounts of the added mucoproteins remained in the DNA fraction, but none of the control protein, bovine serum albumin, remained with the DNA. Further characterization of the mucoproteins by hydroxyapatite chromatography indicated that a certain population of these proteins survived the DNA purification procedures. These data indicate that the association of omeprazole with DNA reported by Phillips et al. most probably is explained by binding of omeprazole to mucous glycoproteins (or other proteins present in the GI tract) that selectively survive DNA purification protocols.

Revalidation of the in vitro alkaline elution/rat hepatocyte assay for DNA damage: improved criteria for assessment of cytotoxicity and genotoxicity and results for 81 compounds.

The in vitro alkaline elution/rat hepatocyte assay is a sensitive assay for genotoxicity, measured as DNA strand breaks induced in primary cultures of rat hepatocytes after 3-h treatments with test compounds. Since DNA degradation can be rapid and extensive in dead and/or dying cells, the original criteria for a positive result in the assay were that a compound induce a 3.0-fold or greater increase in the elution slope (for the terminal phase of alkaline elution from 3 to 9 h) in the absence of significant cytotoxicity (defined as relative cell viability of less than 70% by trypan blue dye exclusion; TBDE). Recently we have shown that false-positive results can still be obtained due to cytotoxicity when loss of membrane integrity is a late event in toxic cell death relative to the induction of endonucleolytic DNA degradation. To improve the ability of the assay to discriminate between genotoxic vs. cytotoxic effects of chemicals, we have evaluated additional assays of cytotoxicity including cell adenosine triphosphate (ATP) and potassium (K+) content, tetrazolium dye reduction (MTT), TBDE after a further 3-h recovery incubation without test chemicals (delayed toxicity), cell blebbing and endonucleolytic DNA degradation (double-strand breaks; DSBs) assessed by pulsed-field gel electrophoresis (PFGE). We have also evaluated 2 parameters derived from the elution data which can indicate extensive, cytotoxicity-induced DNA degradation: the fraction of the DNA recovered in the neutral lysis/rinse fraction and the gamma-intercept of the extrapolation of the 3-9-h segment of the elution curve. Twenty-eight rodent non-carcinogens that are negative (or inconclusive) in the Ames assay with no, or limited, other evidence of genotoxicity, and 33 genotoxins, most of which are also carcinogens, were evaluated. The results showed that DNA degradation as measured by a 1-h PACE (Programmed Autonomously Controlled Electrodes)/PFGE assay was a sensitive indicator of cytotoxicity which correlated well with results of the other cytotoxicity indicators. The delayed TBDE (after a 3-h recovery), intracellular potassium and ATP assays as well as the gamma-intercept parameter were also shown to be sensitive and in some cases complementary measures of cytotoxicity. Using new criteria based on these data of an induced slope (treatment slope-negative control slope) of 0.020 for the 3- to 9-h elution period and cytotoxicity limits of 70% relative viability for the delayed TBDE assay and 50% for intracellular ATP content, the assay scores the genotoxicity of these 61 reference compounds with an overall accuracy of 92%. Test results using these new criteria are provided for an additional 20 compounds (5 non-genotoxic carcinogens and 15 compounds whose genotoxic and carcinogenic potential are unknown or equivocal).

Transgenic tumor models for carcinogen identification: the heterozygous Trp53-deficient and RasH2 mouse lines.

Genetically altered mouse models (GAMM) for human cancers have been critical to the investigation and characterization of oncogene and tumor suppressor gene expression and function and the associated cancer phenotype. Similarly, several of the mouse models with defined genetic alterations have shown promise for identification of potential human carcinogens and investigation of mechanisms of carcinogen-gene interactions and tumorigenesis. In particular, both the B6.129N5-Trp53 mouse, heterozygous for a p53 null allele, and the CB6F1-RasH2 mouse, hemizygous for the human H-ras transgene, have been extensively investigated. Using 26-week exposure protocols at or approaching the maximum tolerated dose, the summary results to date indicate the potential for GAMM to identify and, possibly, classify chemicals of potential risk to humans using short-term carcinogenicity experiments. This IWGT session focused on: (1) the development of recommendations for genetic/molecular characterization required in animals, tissues, and tumors before and after treatment for identification of presumptive human carcinogens based on the current state of knowledge, (2) identification of data gaps in our current state of knowledge, and (3) development of recommendations for research strategies for further development of our knowledge base of these particular models. By optimization of protocols and identification of significant outcomes and responses to chemical exposure in appropriate short-term mechanism-based genetically altered rodent models, strategies for prevention and intervention may be developed and employed to the benefit of public health.

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay.

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed an increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequence of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: comparison with the alkaline sucrose gradient centrifugation technique.

An in vivo-in vitro alkaline DNA unwinding assay for single-strand breaks and/or alkali-labile lesions in hepatic DNA is described. The assay involves isolation of hepatic nuclei from mice, alkaline denaturation and unwinding of hepatic DNA, separation of single- and double-stranded DNA by hydroxylapatite batch chromatography, and quantitation of DNA in chromatographic fractions by a fluorometric assay. The method allows for the sensitive detection of hepatic DNA damage following in vivo administration of xenobiotics. Using this procedure, DNA fragmentation was demonstrated in alkali after administration of as little as 0.5 mg/kg of N-nitrosodimethylamine (DMN) to male B6C3F1 mice. A comparison of this technique with a similar alkaline sucrose density gradient centrifugation assay demonstrated comparable limits of sensitivity for the two procedures.

An investigation of the role of microsomal oxidative metabolism in the in vivo genotoxicity of 1,2-dichloroethane.

In vitro studies have demonstrated that two different metabolic pathways, glutathione conjugation mediated by the glutathione S-transferases and microsomal oxidation, may be involved in the genotoxicity and carcinogenicity of 1,2-dichloroethane (DCE). To evaluate the importance of microsomal oxidative metabolism in the bioactivation of DCE in vivo, male B6C3F1 mice were pretreated with piperonyl butoxide (PIB), an inhibitor of microsomal oxidative metabolism, and the effect of this pretreatment on the extent of hepatic DNA damage produced by DCE was determined 4 hr after DCE administration. The in vivo genotoxicity of 2-chloroethanol, a product of the microsomal oxidative metabolism of DCE, was also investigated. Hepatic DNA damage was measured with a sensitive, alkaline DNA unwinding assay for the presence of single-strand breaks and alkali-labile lesions in DNA. Pretreatment of mice with PIB to inhibit microsomal oxidative metabolism significantly potentiated the hepatic DNA damage observed 4 hr after a single, 200-mg/kg, ip dose of DCE. Treatment of mice with single, ip doses of 2-chloroethanol as high as 1.2 mmol/kg failed to produce any evidence of single-strand breaks and/or alkali-labile lesions in hepatic DNA. When diethyl maleate (DEM) was used to deplete hepatic glutathione levels prior to administration of 2-chloroethanol, the acute hepatotoxicity of 2-chloroethanol was potentiated but again there was no evidence of hepatic damage. These results indicate that microsomal, oxidative metabolism of DCE to 2-chloroethanol and/or 2 chloroacetaldehyde is not responsible for the hepatic DNA damage observed in these studies after DCE administration.

Comparative in vivo genotoxicity and acute hepatotoxicity of three 1,2-dihaloethanes.

Hepatic DNA damage was demonstrated by alkaline DNA unwinding/hydroxylapatite batch chromatography in male B6C3F1 mice treated with non-necrogenic doses of 1,2-dichloroethane, 1-bromo-2-chloroethane, and 1,2-dibromoethane. Intraperitoneal administration of 0.5 mmol/kg of 1-bromo-2-chloroethane and 1,2-dibromoethane produced similar levels of DNA damage. A 4-fold higher dose of 1,2-dichloroethane (2.0 mmol/kg) was required to produce a comparable effect.

The nature of the heterozygous Trp53 knockout model for identification of mutagenic carcinogens.

The heterozygous Trp53 null allele C57BL/6 (N5) mouse is susceptible to the rapid development of neoplasia by mutagenic carcinogens relative to control strains. This mouse model of chemical carcinogenesis demonstrates 1) dose-related rapid induction of tumors (26 wks), 2) multiple sites of carcinogen-specific tissue susceptibility, and 3) carcinogen-induced loss of heterozygosity involving the Trp53 wild-type allele or a p53 haploinsufficiency permitting mutation of other critical protooncogenes and/or inactivation of tumor suppressor genes driving tumorigenesis. Demonstration of mutation or loss of heterozygosity involving the Trp53 locus is consistent with a common finding in human cancers and supports extrapolation between rodents and humans. Using diverse experimental protocols, almost all mutagenic rodent carcinogens (including all mutagens that are carcinogenic to humans), but not nonmutagenic rodent carcinogens, induce tumors within 26 weeks of continuous exposure. These characteristics and results indicate that the mouse heterozygous for the Trp53 null allele may be of significant use for the prospective identification of mutagenic carcinogens of potential risk to human health.

Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.