Prenatal toxicity and lack of carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) following transplacental exposure.

Accumulating evidence from human and experimental animal studies indicates that consumption of heterocyclic amines (HA), derived from cooked meat and fish, may be associated with an increased incidence of cancer. Experiments were initiated to assess the role of one of these compounds, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as a potential transplacental carcinogen, as well as to evaluate whether in utero exposure to IQ results in the induction of fetal cytochrome P4501A1 (Cyp1a1), P4501B1 (Cyp1b1), and/or glutathione S-transferase (GST). Inducible, or responsive, backcrossed fetuses resulting from a cross between congenic C57BL/6 (Ah(d)Ah(d)) nonresponsive female mice and C57BL/6 (Ah(b)Ah(b)) responsive male mice were transplacentally exposed to olive oil or 6.25, 12.5, or 25 mg/kg of IQ on day 17 of gestation. No macroscopically or microscopically visible liver, lung, or colon tumors were found in the transplacentally treated offspring by one year after birth. Ethoxyresorufin O-deethylase (EROD) and 1-chloro-2,4-dinitrobenzene assays were performed to evaluate whether transplacental exposure to IQ results in the induction of fetal Cyp1a1 and GST, respectively, in lung and liver tissues. Results showed levels of EROD and GST activity in tissues of IQ-treated mice to be very close, if not identical, to those of mice treated with olive oil. Similarly, ribonuclease protection assay data showed that the levels of Cyp1a1 and Cyp1b1 RNA in tissues of IQ-treated mice were not significantly different from those of oil-treated controls. Previous studies have shown that the developing organism expresses very low levels of Cyp1a2. Thus, in utero exposure to IQ does not lead to induction of Cyp1a1, Cyp1a2, or Cyp1b1 in the fetal compartment, thereby maintaining the low levels of these activating enzymes in the developing organism. Taken together, these data imply that, at least under the conditions employed for these experiments, IQ may not play an important role in transplacentally induced tumorigenesis.

Differential sensitivity to lung tumorigenesis following transplacental exposure of mice to polycyclic hydrocarbons, heterocyclic amines, and lung tumor promoters.

Research conducted by this laboratory over the past decade has demonstrated the high susceptibility of the fetus to lung tumor formation following in utero exposure of the resistant C57BL/6 and DBA/2N strains of mice to 3-methylcholanthrene (MC). In this review, we describe our more recent studies on the effects of MC and cotreatment with the lung tumor promoter, butylated hydroxytoluene (BHT), on lung tumor formation in the intermediately susceptible BALB/c strain of mice, and the determination of the potential carcinogenicity of the heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in resistant mouse strains. BALB/c mice showed a similar incidence of lung tumors, both in terms of percentage of mice with tumors and number of tumors per mouse, as found in the resistant [D2 x B6D2F1]F2 mice. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung lesions compared with an incidence of 79% in [D2 x B6D2F1]F2 mice. BALB/c lung lesions demonstrated a similar association of Ki-ras mutations with tumor stage. Interestingly, a strain-dependent difference was observed in the mutational spectrum, where 62% and 38% of the lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast with the 16% and 84% incidences observed in [D2 x B6D2F1]F2 mice. BHT had no statistically significant effect on tumor incidence, multiplicity, or Ki-ras mutational spectrum in BALB/c mice treated in utero with MC, although a trend toward increased tumor multiplicity was observed. Finally, experiments initiated to assess the transplacental carcinogenicity of IQ in D2B6F1 mice demonstrated that 1 year after birth, no macroscopically or microscopically visible liver, lung, or colon tumors were found in the transplacentally treated offspring, nor was induction of Cyp1a1, Cyp1b1, or glutathione S-transferases (GSTs) in fetal lung and liver tissues observed. This implies that at least under these experimental conditions, IQ may not be an important transplacental carcinogen. Overall, these data demonstrate that mutagenic damage to Ki-ras is a critical early event mediating murine lung tumorigenesis in both sensitive and resistant strains. Strain-dependent differences in the Ki-ras mutational spectrum may be associated with their differential susceptibility to lung tumor initiation.

Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and post-natally exposed to butylated hydroxytoluene.

The carcinogenic effects of in utero exposure to 3-methylcholanthrene (MC) have been demonstrated in the tumor-resistant C57BL/6 (B6) and DBA (D2) strains of mice. In this study, we determined the effects of in utero exposure to MC in BALB/c mice, a strain which demonstrates greater susceptibility to lung tumor induction, and compared our findings with those previously found in [D2xB6D2F(1)]F(2) mice. In addition, we assessed the molecular pathogenesis of the chemically induced tumors and examined the effects of the putative lung tumor promoter butylated hydroxytoluene (BHT) in BALB/c mice. BALB/c mice were treated on day 17 of gestation with 5, 15 or 45 mg/kg MC and 6 weeks after birth with BHT for 6 consecutive weeks. Mice were killed at 6 months of age. Ki-ras, p16Ink4a and p19ARF gene loci were amplified from paraffin-embedded lung tumor tissue and screened for the presence of point mutations via allele-specific oligonucleotide hybridization and single strand conformation polymorphism (SSCP) analyses. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung tumors, with 33% (2/6) of the hyperplasias, 58% (10/19) of the adenomas and 73% (8/11) of the carcinomas exhibiting point mutations at this gene locus. Similar incidences of Ki-ras mutations were previously found following transplacental exposure of [D2xB6D2F(1)]F(2) mice to MC and treatment of adult A/J mice with urethane. Interestingly, a strain-dependent difference was observed in the mutational spectrum. Sixty-two and 38% of the lung lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast to the 13 and 84% incidences previously observed in [D2xB6D2F(1)]F(2) mice. SSCP analysis of the tumor suppressor gene p16Ink4a showed a 6% incidence of point mutations, consistent with that found in [D2xB6D2F(1)]F(2) mice. No mutations were found in exon 1beta of the p19ARF gene of either strain. BHT, a lung tumor promoter in adult mice, had no statistically significant effects on either tumor incidence, tumor multiplicity or the mutational spectrum produced in the Ki-ras gene by in utero MC treatment. However, though not significant, there was an observable trend in increased tumor multiplicity in mice co-treated with BHT. These data demonstrate the transplacental carcinogenic effect of MC in BALB/c mice and show that mutagenic damage to Ki-ras is a critical early event mediating murine lung tumorigenesis in both the tumor-sensitive and tumor-resistant strains. Unlike what occurs when adult BALB/c mice are treated with MC, BHT does not appear to significantly promote the formation of lung tumors following transplacental exposure to MC, possibly due to the rapid growth and cell proliferation in the developing organism. Strain-dependent differences in the Ki-ras mutational spectrum may be associated with their differential susceptibility to lung tumor initiation.

Induction of mutations in Ki-ras and INK4a in liver tumors of mice exposed in utero to 3-methylcholanthrene.

An understanding of the basic mechanisms responsible for the pathogenesis of liver neoplasms is needed in order to develop better therapeutic strategies. The present study utilized a pharmacogenetic mouse model to assess the role of cytochrome P4501A1 (Cyp1a1) in modulating genetic damage to oncogenic and tumor suppressor loci following in utero exposure to the polycyclic aromatic hydrocarbon, 3-methylcholanthrene (MC). Analysis of the Ha-ras, Ki-ras, INK4a and p53 genes was carried out with lysates from paraffin-embedded liver tissue from transplacentally-treated mice. The lysates were subjected to DNA amplification by the PCR technique followed by allele-specific oligonucleotide hybridization screening and SSCP analysis. All of the 26 neoplasms screened (23 hepatocellular carcinomas, two hepatocellular adenomas and one sarcoma) exhibited a GGC-->CGC (GLY13-->ARG13) transversion at the Ki-ras gene locus. None of the tumors had Ki-ras mutations at codon 12 of exon 1. Approximately 12% (3/26) of the liver tumors exhibited point mutations in exon 1 of the INK4a gene, with each of the three tumors exhibiting two point mutations. Analysis of exon 2 of the INK4a gene showed the presence of a CCG-->CTG (PRO73-->LEU73) transition in two of the 26 neoplasms. No mutations were found in exons 1 or 2 of the Ha-ras gene, or in exons 5-8 of the p53 gene. Analysis of tumor RNAs showed overexpression of Ha-ras, cip1 and c-jun in approximately 38% of the liver tumor samples. The results of this study suggest that mutagenic damage to oncogenes and tumor suppressor genes may be critical factors in mediating transplacentally-induced liver tumorigenesis. The fact that Ki-ras mutations were found in all of the tumors suggests that mutation at this gene locus may be an early event in liver tumor pathogenesis, while mutation in tumor suppressor genes may occur later during tumor progression. These combined results are consistent with the pathogenesis of cancer in humans.