Anthracene Treatment Research Articles

Apigenin Prevents Development of Medroxyprogesterone Acetate-Accelerated 7,12-Dimethylbenz(a)anthracene-Induced Mammary Tumors in Sprague–Dawley Rats

The use of progestins as a component of hormone replacement therapy has been linked to an increase in breast cancer risk in postmenopausal women. We have previously shown that medroxyprogesterone acetate (MPA), a commonly administered synthetic progestin, increases production of the potent angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells, leading to the development of new blood vessels and tumor growth. We sought to identify nontoxic chemicals that would inhibit progestin-induced tumorigenesis. We used a recently developed progestin-dependent mammary cancer model in which tumors are induced in Sprague-Dawley rats by 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The flavonoid apigenin, which we previously found to inhibit progestin-dependent VEGF synthesis in human breast cancer cells in vitro, significantly delayed the development of, and decreased the incidence and multiplicity of, MPA-accelerated DMBA-induced mammary tumors in this animal model. Whereas apigenin decreased the occurrence of such tumors, it did not block MPA-induced intraductal and lobular epithelial cell hyperplasia in the mammary tissue. Apigenin blocked MPA-dependent increases in VEGF, and suppressed VEGF receptor-2 (VEGFR-2) but not VEGFR-1 in regions of hyperplasia. No differences were observed in estrogen or progesterone receptor (ER/PR) levels, or the number of estrogen receptor-positive cells, within the mammary gland of MPA-treated animals administered apigenin, MPA-treated animals, and placebo treated animals. However, the number of progesterone receptor-positive cells was reduced in animals treated with MPA or MPA and apigenin compared with those treated with placebo. These findings suggest that apigenin has important chemopreventive properties for those breast cancers that develop in response to progestins.

Modulation of biomarkers related to tumor initiation and promotion in mouse skin by a natural β-glucuronidase inhibitor and its precursors

Carcinogen-mediated labilization of lysosomal enzymes such as β-glucuronidase (βG) is often associated with the general process of inflammation. Therefore, the primary goal of this study was to demonstrate that exposing the skin of SENCAR mice to the natural βG inhibitor D-glucaro-1,4-lactone (1,4-GL) and its precursor D-glucuronic acid-γ-lactone (GUL), prior to and during 7,12-dimethylbenz[α]anthracene (DMBA) treatment inhibits not only epidermal hyperplasia but also inflammation in the mouse skin complete carcinogenesis model, i.e., the 4-week inflammatory-hyperplasia assay. Topical administration of 1,4-GL or GUL prior to repetitive, high-dose DMBA treatment markedly and in a dose-related manner inhibited DMBA-induced epidermal hyperplasia (i.e., up to 57%). DMBA-mediated Ha-ras mutations in codon61 were reduced by up to 78% by 1,4-GL. DMBA-induced inflammation, as measured by dermal leukocyte counts and immunologically, was inhibited by up to 37% by topical 1,4-GL but not by GUL. The inhibition of cellular proliferation and inflammation coincided with the inhibition of βG expression. Thus, the present study suggests that in the DMBA-induced complete skin carcinogenesis model, 1,4-GL when applied topically had both anti-proliferative properties as well as anti-inflammatory properties, whereas GUL had only anti-proliferative when applied topically. However, the number of inflammatory cells in the dermal portion of the skin of mice was significantly reduced by dietary treatment of GUL, whereas both topical and dietary treatments with 1,4-GL were very effective.

Aggressive mammary carcinoma progression in Nrf2 knockout mice treated with 7,12-dimethylbenz[a]anthracene

BackgroundActivation of nuclear factor erythroid 2-related factor (Nrf2), which belongs to the basic leucine zipper transcription factor family, is a strategy for cancer chemopreventive phytochemicals. It is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1 and peroxiredoxin 1, by activating the antioxidant response element (ARE). We hypothesized that (1) the citrus coumarin auraptene may suppress premalignant mammary lesions via activation of Nrf2/ARE, and (2) that Nrf2 knockout (KO) mice would be more susceptible to mammary carcinogenesis.MethodsPremalignant lesions and mammary carcinomas were induced by medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene treatment. The 10-week pre-malignant study was performed in which 8 groups of 10 each female wild-type (WT) and KO mice were fed either control diet or diets containing auraptene (500 ppm). A carcinogenesis study was also conducted in KO vs. WT mice (n = 30-34). Comparisons between groups were evaluated using ANOVA and Kaplan-Meier Survival statistics, and the Mann-Whitney U-test.ResultsAll mice treated with carcinogen exhibited premalignant lesions but there were no differences by genotype or diet. In the KO mice, there was a dramatic increase in mammary carcinoma growth rate, size, and weight. Although there was no difference in overall survival, the KO mice had significantly lower mammary tumor-free survival. Also, in the KO mammary carcinomas, the active forms of NF-κB and β-catenin were increased ~2-fold whereas no differences in oxidized proteins were observed. Many other tumors were observed, including lymphomas. Interestingly, the incidences of lung adenomas in the KO mice were significantly higher than in the WT mice.ConclusionsWe report, for the first time, that there was no apparent difference in the formation of premalignant lesions, but rather, the KO mice exhibited rapid, aggressive mammary carcinoma progression.

ChemInform Abstract: A General and Simple Route to the Synthesis of Triptycenes.

A general and simple route to the synthesis of the triptycene derivatives 5,12-dihydro-5,12[1',2']-benzenonaphthacene (1), 5,14-dihydro-5,14[1',2']-benzenopentacene (2) and 5,16-dihydro-5,16[1',2']-benzenohexacene (3) is described. It involves the cycloaddition of appropriate quinone to anthracene and subsequent treatment of the resulting adduct with lithium aluminium hydride, followed by p-toluenesulfonyl chloride in pyridine

425 IGFBP-3 knockout mice develop earlier mammary tumours following dimethylbenz[a]anthracene treatment

425 IGFBP-3 knockout mice develop earlier mammary tumours following dimethylbenz[a]anthracene treatment

Targeted disruption of Bcl‐xL in mouse keratinocytes inhibits both UVB‐ and chemically induced skin carcinogenesis

Bcl-x(L) is one of several antiapoptotic proteins regulated by signal transducer and activator of transcription 3 (Stat3). We have recently shown that Stat3 is required for chemically induced and ultraviolet B (UVB)-induced skin carcinogenesis. In this study, the functional role of Bcl-x(L) in skin carcinogenesis was investigated using skin-specific Bcl-x(L)-deficient mice. In this model, Bcl-x(L) expression is disrupted in the basal compartment of mouse epidermis using the bovine keratin 5 (K5) promoter to drive expression of Cre recombinase (K5.Cre x Bcl-x(L) (fl/fl) mice). A significant increase in apoptosis induced by either UVB irradiation or 7,12-dimethylbenz[a]anthracene (DMBA) treatment was observed in the epidermis of Bcl-x(L)-deficient mice. Furthermore, an increase in apoptotic cells was noted in hair follicle keratinocytes, including those located in the bulge region. Cell proliferation was not affected by Bcl-x(L) deficiency following exposure to either UVB or 12-O-tetradecanoylphorbol-13-acetate (TPA). Bcl-x(L)-deficient mice were more resistant than wild-type controls to skin tumor development with delayed onset and reduced number of tumors using either UVB or the DMBA/TPA two-stage regimen. Moreover, Bcl-2, Mcl-1, and survivin protein levels were increased in the epidermis of Bcl-x(L)-deficient mice in the absence of stimuli. Furthermore, levels of these antiapoptotic proteins were also high in skin tumors from Bcl-x(L)-deficient mice that developed in response to either UVB or two-stage carcinogenesis protocols. Collectively, these studies demonstrate that Bcl-x(L) plays a role early in skin carcinogenesis through its anti-apoptotic functions to enhance survival of keratinocytes, including bulge region keratinocyte stem cells, following DNA damage.

Aryl hydrocarbon receptor polymorphism modulates DMBA‐induced inflammation and carcinogenesis in phenotypically selected mice

We tested the role of aryl hydrocarbon receptor (Ahr) gene polymorphism in the inflammatory response and in skin and lung tumorigenesis in 2 lines of mice phenotypically selected for maximum or minimum acute inflammatory reaction (AIRmax and AIRmin, respectively). Following 7,12-dimethylbenz[a]anthracene (DMBA) treatment, AIRmin but not AIRmax mice showed early skin reactions and eventually developed malignant skin tumors and lung adenocarcinomas. In skin tissue, transcript levels of IL1beta, Tnf, Il6, Tgfbeta1 and Cyp1b1 genes were upregulated in AIRmin but not AIRmax mice, consistent with the inflammatory responses to the carcinogen. These findings appeared to be related to the homozygosity status of the Ahr functional A375V polymorphism, which influences the binding capability of the receptor for DMBA: the 375A allele, encoding the high-affinity ligand-binding receptor (Ahr(b1)), segregated in AIRmin mice, whereas AIRmax mice carried the 375V, corresponding to the low-affinity binding receptor (Ahr(d)), to DMBA. The differential segregation of Ahr functional Ahr(d)versus Ahr(b1) alleles in AIRmax and AIRmin suggests a role for the Ahr gene in the control of inflammatory responsiveness and tumor development of these mouse lines.

In vitro evaluation of the oxidative stress and genotoxic potentials of anthracene on mycorrhizal chicory roots

Most polycyclic aromatic hydrocarbons (PAHs) are ubiquitous natural and/or anthropogenic pollutants that have adverse effects on the human health and the environment. Little is known about their potential effects on higher plants and especially the mycorrhizal ones. In order to improve the understanding of the effects of PAHs on the partners of the endomycorrhizal symbiotic association, the oxidative stress and genotoxicity potentials of anthracene was investigated in Glomus intraradices-colonized and non-colonized transformed Cichorium intybus roots grown in in vitro cultures. Toxic effects of anthracene on the arbuscular mycorrhizal (AM) fungi was showed in the presence of increasing concentrations of anthracene (6.25–50 mg L −1), as compared to the control culture. The extraradical hyphae density and the sporulation intensity were drastically restricted in the presence of anthracene. The colonization of chicory roots was significantly affected. Chicory root lengths were reduced by 13% when grown on anthracene containing medium. The level of lipid peroxidation, as assessed by malondialdehyde (MDA) production, was markedly increased in colonized roots under anthracene treatment. The genomic alteration, as studied by the 8-hydroxy-2′-desoxyguanosine (8-OHdG) oxidative adduct formation, increased by about 2 folds in exposed roots as compared to the control. Significant decreases were detected in the MDA and 8-OHdG concentrations in colonized roots as compared to non-colonized roots exposed to anthracene. Taken together, these results suggested that endomycorrhization is able to give protection to the chicory roots against the lipid peroxidation and the genotoxicity of anthracene by preventing formation of MDA and 8-OHdG.

Effects of obesity and 7,12‐dimethylbenz(a)anthracene (DMBA) treatment on hepatic CYP 1A1 expression in ovariectomized obese Zucker rats

There is a link between obesity and increased risk of breast cancer among post‐menopausal women. Recently, we have shown that obesity increases the rate of the DMBA‐induced mammary tumor development in intact and ovariectomized Zucker rats. Several data suggest that DMBA require metabolic activation in order to exert their carcinogenic effects. The objective of this study was to investigate the effect of obesity on hepatic expression of CYP1A1 following treatment in obese and lean ovariectomized female Zucker rats. Forty day‐old, ovariectomized obese (n=20) and lean (n=20), female Zucker rats were kept on AIN‐93 G diet and 10 days later were orally gavaged with either with sesame oil (control) or with 65 mg/kg DMBA in sesame oil and were sacrificed 24 hrs post‐DMBA treatment. Liver microsomes were prepared and CYP 1A1 expression were measured by Western blotting using goat anti‐mouse CYP 1A1. DMBA treatment significantly (p<0.001) increased expression of CYP 1A1 in lean and obese ovariectomized rats compared to control group. Obese rats had higher basal CYP 1A1 expression compared to lean rats. These data suggest that obesity can increase expression of enzymes that are responsible for DMBA metabolism using ovariectomized zucker rat model. Also, DMBA treatment will increase the expression of CYP 1A1. (Supported by Susan G. Komen Foundation and ABI to RH).

Effect of a plant-originated natural compound, CoD™ extract, on c-myc, Ha-ras and p53 gene expression in short-term animal experiments

Several plant-originated natural compounds were shown to exhibit chemopreventive effect in carcinogenesis. In our studies, we have investigated CoD™ extract, a complex plant extract made from several tropical plants with possible cancer preventive effect in animal experiments. c-myc, p53 and Ha-ras gene expressions were investigated 24, 48 and 72 h after CoD™ extract and dimethylbenz(a)-anthracene (DMBA) treatment in CBA/Ca(H-2 K ) mice. CoD™ extract administered together with DMBA diminished c-myc, p53 and Ha-ras gene expressions both in the 24 and 48 h experiments, but not in the 72 h experiments. Further in vivo and human studies are needed to clarify the possible role of CoD™ extract in the prevention of tumour formation after carcinogenic exposures.

Enhanced Susceptibility to Chemical Induction of Ovarian Tumors in Mice with a Germ Line p53 Mutation

Mice with a germ line p53 mutation (p53(Ala135Val/wt)) display increased susceptibility to lung, skin, and colon carcinogenesis. Here, we show that p53(Ala135Val/wt) mice developed ovarian tumors significantly more rapidly than their wild-type littermates after 7,12-dimethylbenz(a)anthracene (DMBA) treatment. Approximately 50% of the ovarian tumors in p53(wt/wt) mice and 23% in p53(Ala135Val/wt) mice are adenocarcinomas and the remaining tumors were adenocarcinoma mixed with sarcoma or ovarian sarcomas. All of the p53(Ala135Val/wt) mice had died of ovarian tumors 25 weeks after the initial DMBA treatment, whereas >50% of p53(wt/wt) mice were still alive. These mice not only have a shortened tumor latency but also closely resemble a subset of human ovarian tumors containing the p53 mutation. Microarray and GenMAPP analyses revealed that the mutant p53 (Ala135Val) affected several cellular processes, including the cell cycle, apoptosis, and Wnt pathways. These findings indicate that a germ line p53 mutation significantly enhanced DMBA-induced ovarian tumor development and progression.

RelB/p52 NF-κB Complexes Rescue an Early Delay in Mammary Gland Development in Transgenic Mice with Targeted Superrepressor IκB-α Expression and Promote Carcinogenesis of the Mammary Gland

Classical NF-kappaB (p65/p50) transcription factors display dynamic induction in the mammary gland during pregnancy. To further elucidate the role of NF-kappaB factors in breast development, we generated a transgenic mouse expressing the IkappaB-alpha S32/36A superrepressor (SR) protein under control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. A transient delay in mammary ductal branching was observed in MMTV-SR-IkappaB-alpha mice early during pregnancy at day 5.5 (d5.5) and d7.5; however, development recovered by mid- to late pregnancy (d14.5). Recovery correlated with induction of nuclear cyclin D1 and RelB/p52 NF-kappaB complexes. RelB/p52 complexes induced cyclin D1 and c-myc promoter activities and failed in electrophoretic mobility shift assay to interact with IkappaB-alpha-glutathione S-transferase, indicating that their weak interaction with IkappaB-alpha can account for the observed recovery of mammary gland development. Activation of IKKalpha and NF-kappaB-inducing kinase was detected by d5.5, implicating the alternative NF-kappaB signaling pathway in RelB/p52 induction. Constitutively active IKKalpha induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment displayed increased RelB/p52 activity. Inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar. These results implicate RelB/p52 complexes in mammary gland development and carcinogenesis.

Mice Expressing a Mammary Gland–Specific R270H Mutation in the p53 Tumor Suppressor Gene Mimic Human Breast Cancer Development

The tumor suppressor gene p53 has an apparent role in breast tumor development in humans, as approximately 30% of sporadic tumors acquire p53 mutations and Li-Fraumeni syndrome patients carrying germ line p53 mutations frequently develop breast tumors at early age. In the present study, conditional expression of a targeted mutation is used to analyze the role of the human R273H tumor-associated hotspot mutation in p53 in mammary gland tumorigenesis. Heterozygous p53(R270H/+)WAPCre mice (with mammary gland-specific expression of the p53.R270H mutation, equivalent to human R273H, at physiologic levels) develop mammary tumors at high frequency, indicating that the R270H mutation predisposes for mammary gland tumor development and acts in a dominant-negative manner in early stages of tumorigenesis. Spontaneous tumor development in these mice is further accelerated by 7,12-dimethylbenz(a)anthracene (DMBA) treatment at young age. The majority of spontaneous and DMBA-induced carcinomas and sarcomas from p53(R270H/+)WAPCre mice is estrogen receptor alpha positive, and expression profiles of genes also implicated in human breast cancer appear similarly altered. As such, p53(R270H/+)WAPCre mice provide a well-suited model system to study the role of p53 in breast tumorigenesis and the responsiveness of mammary gland tumors to chemotherapeutics.

7,12-Dimethylbenzanthracene-dependent transcriptional regulation of adenomatous polyposis coli (APC) gene expression in normal breast epithelial cells is mediated by GC-box binding protein Sp3

In the present investigation, we have examined the transcriptional regulation of adenomatous polyposis coli (APC) gene expression in the spontaneously immortalized human normal breast epithelial cell line, MCF10A, in response to carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The APC mRNA levels and the APC gene's promoter (pAPCP) activity were increased in MCF10A cells after treatment with DMBA. A sequential deletion analysis and site-directed mutagenesis of the pAPCP promoter revealed that the DMBA response is mediated through a GC-box element. Also, the GC-box binding agent mithramycin A, which prevents binding of proteins to the GC-box region, abolished DMBA-mediated increase of the pAPCP promoter activity. The specificity of the proteins binding to the GC-box region was characterized by gel-shift analysis. An increased binding of the GC-box binding proteins was observed in the gel-shift analysis with nuclear extracts from DMBA-treated MCF10A cells, which corresponded to the increased levels of Sp1 and Sp3 proteins. However, a super-shift of the DNA-protein complexes was observed with only anti-Sp3 antibody. Based on the chromatin-immunoprecipitation assay results, the Sp3 appeared to be a genuine protein binding to the GC-box site of the pAPCP promoter. In RNA interference experiments, in which the Sp3 expression was knocked down, the DMBA response on the pAPCP promoter activity was reduced, suggesting that the binding of Sp3 to the GC-box site is critical for DMBA-induced pAPCP promoter activity. From these results we conclude that the increased pAPCP promoter activity in the MCF10A cell line in response to DMBA treatment is mediated by Sp3.

Identification of an X-linked locus modifying mouse skin tumor susceptibility

The enhancing effect of overexpression of an ornithine decarboxylase (Odc) transgene on skin tumor susceptibility can be modified by genetic loci present in several inbred mouse strains. The BALB/cJ strain is among the most resistant strains so far examined; tumor multiplicity following 7,12-dimethylbenz(a)anthracene (DMBA) treatment is reduced by 90% when the K6/ODC transgene is expressed on a BALB/cJ background versus the susceptible C57BL/6J background. Further, transgenic BALB/cJ males developed more tumors than females, indicating the presence of sex-dependent modifier pathway. Analysis of 263 F2 intercross mice revealed significant linkage of markers on the X chromosome to tumor multiplicity. This analyses as well as a similar genome-wide scan of 136 backcross mice found evidence for other modifier loci on chromosomes 4, 6, and 17. Identification of these modifier genes should reveal the effector pathways responsive to Odc overexpression that mediate susceptibility to skin tumorigenesis.

Effect of Onosma echioides on DMBA/croton oil mediated carcinogenic response, hyperproliferation and oxidative damage in murine skin

The current study unveils the effect of O. echioides extract on two-stage skin carcinogenesis and on tumor promoter induced markers and oxidative stress in Swiss mice. Treatment of dorsal shaven cutaneous portions of the mice with single topical application of benzoyl peroxide (BPO) followed by exposure to ultraviolet B (UVB) radiation induced significant oxidative stress and elevated the marker parameters of tumor promotion. Similar effects were observed with 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment. Pretreatment of O. echioides extract (5 mg & 10 mg/Kg b.wt) in both the studies with BPO+UVB and TPA restored the levels of reduced glutathione (GSH) and cellular protective enzymes (p < 0.05). Concomitantly, malondialdehyde (MDA) formation and hydrogen peroxide (H 2O 2) content were also reduced significantly (p < 0.05) at both the doses. The promotion parameters tested (ornithine decarboxylase activity and DNA synthesis) were also significantly suppressed (p < 0.05). Thereafter, we proceeded with studies on mouse skin carcinogenesis. After ten days of 7,12-dimethylbenz(a)anthracene (DMBA) treatment, twice-weekly applications of croton oil for 20 weeks resulted in 100% incidence of tumors in the animals. However, O. echioides showed reduction in the number of tumors/ mouse and percentage of tumor bearing mice at the end of the study. The study was further histologically confirmed. The protective activity of the plant might be due to the two major constituents (alkannins and shikonins) present in the plant. O. echioides is thus, proposed to be helpful in prevention of experimental skin carcinogenesis.

Overexpression of the Colony-Stimulating Factor (CSF-1) and/or Its Receptor c-fms in Mammary Glands of Transgenic Mice Results in Hyperplasia and Tumor Formation

A number of recent studies have suggested that the colony-stimulating factor (CSF-1) and its receptor c-fms may be involved in the development of mammary glands during lactation and breast cancer. To study the role of CSF-1 or its receptor in initiation of mammary tumorigenesis, we have generated two independent lines of transgenic mice that overexpress either CSF-1 or c-fms under the control of the mouse mammary tumor virus promoter. Mammary glands of the virgin CSF-1 transgenic mice show increased ductal branching, hyperplasia, dysplasia, and other preneoplastic changes, which are indicative of increased cellular proliferation. Similar changes were also evident in the mammary glands of the c-fms transgenic mice. These changes became more prominent with age and resulted in mammary tumor formation. Moreover, secondary events like dimethylbenz(a)anthracene treatment accelerated mammary tumor formation in these mice. Although the expression of estrogen receptor alpha was not significantly changed in either of the transgenic mouse strains, progesterone receptor levels was higher in both transgenic lines as compared with the nontransgenic littermates. Expression of G1 cyclins was prominently increased in the mammary glands of both the CSF-1 and c-fms transgenic lines, suggesting increased cell cycle progression in these strains. In addition, the proliferation marker proliferating cell nuclear antigen (PCNA) and the mitogen-responsive transcription factor c-jun were also increased as compared with the nontransgenic controls. These findings, along with the histological data, support the hypothesis that CSF-1 and its receptor are involved in the etiology of breast cancer.

Chemical carcinogens induce varying patterns of LOH in mouse T-lymphocytes.

We have shown previously that a wide range of mutagenic carcinogens are capable of inducing loss of heterozygosity (LOH) at the endogenous Aprt locus in mouse splenic lymphocytes. To investigate whether LOH might be caused by a single common mechanism, we set out to determine the extent of LOH by microsatellite analysis along (the Aprt gene containing) mouse chromosome 8. Aprt+/- hybrid B6C3F1 mice were treated with mutagens that induce different classes of DNA lesions, i.e. bulky DNA adducts, DNA methylation, DNA inter-strand crosslinks or DNA strand breaks. Aprt mutant frequencies (MF) in this C57Bl/6-C3H hybrid background were significantly reduced for mitomycin C (MMC) and methylmethanesulfonate (MMS) in comparison with MF in C57Bl/6 background, suggesting either enhanced repair or reduced formation of MMC- or MMS-induced mutagenic lesions in a hybrid B6C3F1 background. In contrast, Aprt MF after dimethylbenz[a]anthracene (DMBA), methylnitrosurea (MNU) and etoposide treatment were similar in both genetic backgrounds. Microsatellite analysis of Aprt mutant clones indicated a dominant role for mitotic recombination (MR) in generating spontaneous, DMBA- and etoposide-induced LOH at APRT: However, over 80% of the MMC-induced Aprt LOH mutants had lost heterozygosity for all markers tested, suggesting that either the crossover points were located close to the centromere or that these mutants arose by chromosome loss and duplication of the remaining chromosome 8. A substantial fraction (40%) of MNU-induced Aprt mutants had lost the wild-type Aprt allele, but had retained heterozygosity at all polymorphic markers tested at chromosome 8 indicating an important role for deletions in LOH formation by MNU. Patterns of MR differed quite dramatically for the various chemical mutagens tested, suggesting different mechanisms to be involved in inducing recombination between homologous chromosomes. In addition, non-random adduct formation and repair between chromosomal regions, i.e. heterochromatin versus euchromatin, may contribute to a non-random distribution of recombinational crossover points.

Effect of calorie restriction on mortality kinetics in inbred strains of mice following 7,12-dimethylbenz[a]anthracene treatment.

Calorie restriction (CR) has long been known to increase longevity and to delay the onset and to decrease the incidence of many age-related disease processes. The mechanism(s) by which these outcomes are attained is unidentified. This experiment was designed to examine whether differences existed in the extent to which various inbred strains of mice respond to CR. This work explored whether carcinogen-treated animals could be used to facilitate this aim by decreasing the time needed to observe differences in mortality kinetics between CR mice and ad libitum (AL) fed controls. Female mice from each of eight strains (A/J, BALB/c, C3H, C57BL/6, DBA/2J, FVB/J, NMRI, and 129/J) were given a single oral dose (65 mg/kg) of the carcinogen 7,12-dimethylbenz[a]anthracene and subsequently fed AL or calorically restricted. Following carcinogen treatment, the spectrum of lesions observed demonstrated genotypic variability, thereby complicating comparison among the inbred strains examined. However, in terms of the magnitude of alteration in mortality kinetics observed, a statistical analysis revealed that differences exist among the various strains of mice in their response.

Importance of DNA repair in carcinogenesis: evidence from transgenic and gene targeting studies

We have generated transgenic mice by introducing copies of the E. coli O 6-methylguanine-DNA methyltransferase gene, ada. Liver extracts from homozygotes demonstrate about three times the control enzyme activity and increase up to about eight-fold can be induced by treatment with zinc, since the metal-responsive metallothionein promoter is attached to the ada gene. Furthermore, studies of liver carcinogenesis in our transgenic mice demonstrated significantly reduced rates of development of hepatocellular tumors after treatment with dimethylnitrosamine or diethylnitrosamine. It is well known that xeroderma pigmentosum (XP) patients are deficient in DNA repair. The availability of XPA (XP group A complementing) knockout mice has enabled us to investigate the functional role of the XPA nucleotide excision repair gene in carcinogenesis in vivo, first using the mouse skin as a model system. XPA−/− mice demonstrated skin ulcers 5–7 days after 7,12-dimethylbenz[a]anthracene (DMBA) treatment and papilloma development within 4 weeks prior to promotion, skin tumor incidence being also much higher than in heterozygous and wild-type mice. Experiments targeting the lung, liver and tongue have also been conducted to answer the question of whether the internal organs of these mice are also susceptible to chemical carcinogens. For lung carcinogenesis, mice were instilled intratracheally with a small dose of benzo[a]pyrene. The pulmonary tumor incidence in XPA−/− mice was significantly higher than in XPA+/− and XPA+/+ mice. XPA−/− mice were also found to be have enhanced sensitivity to aflatoxin B 1 regarding liver tumor induction. In addition, administration of 4-nitroquinoline-1-oxide in drinking water for 50 weeks resulted in tongue tumors only in XPA−/− mice. These studies, thus, provided convincing evidence that XPA mice are also sensitive to carcinogenesis in organs other than the skin.