Tumors and proliferative lesions in adult offspring after maternal exposure to methylarsonous acid during gestation in CD1 mice.

Developmental exposure to inorganic arsenic is carcinogenic in humans and mice, and adult offspring of mice exposed to inorganic arsenic can develop tumors of the lung, liver, adrenal, uterus, and ovary. It has been suggested that methylarsonous acid (MMA3+), a product of the biological methylation of inorganic arsenic, could be a key carcinogenic species. Thus, pregnant CD1 mice were provided drinking water containing MMA3+ at 0 (control), 12.5, or 25 parts per million (ppm) from gestational days 8 to 18. Tumors were assessed in groups of male or female (initial n = 25) offspring up to 2 years of age. In utero treatment had no effect on survival or body weights. Female offspring exhibited increases in total epithelial uterine tumors (control 0%; 12.5 ppm 26%; 25 ppm 30%), oviduct hyperplasia (control 4%; 12.5 ppm 35%; 25 ppm 43%), adrenal cortical adenoma at 25 ppm (control 0%; 12.5 ppm 9%; 25 ppm 26%), and total epithelial ovarian tumors (control 0%; 12.5 ppm 39%; 25 ppm 26%). Male offspring showed dose-related increases in hepatocellular carcinoma (control 0%; 12.5 ppm 12%; 25 ppm 22%), adrenal adenoma (control 0%; 12.5 ppm 28%; 25 ppm 17%), and lung adenocarcinoma (control 17%; 12.5 ppm 44%). Male offspring had unusual testicular lesions, including two rete testis carcinomas, two adenomas, and three interstitial cell tumors. Overall, maternal consumption of MMA3+ during pregnancy in CD1 mice produced some similar proliferative lesions as gestationally applied inorganic arsenic in the offspring during adulthood.

Effects of ascorbic acid on carcinogenicity and acute toxicity of nickel subsulfide, and on tumor transplants growth in gulonolactone oxidase knock-out mice and wild-type C57BL mice.

The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono--lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni3S2), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni3S2 carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni3S2. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni3S2 in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni3S2 carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni3S2 and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies.

Inheritance of susceptibility to induction of nephroblastomas in the Noble rat.

Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53% of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28% and 30%, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46%, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16%). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.

Arsenic exposure in utero and nonepidermal proliferative response in adulthood in Tg.AC mice.

To expand our knowledge on the transplacental carcinogenic potential of inorganic arsenic, pregnant Tg.AC mice received drinking water with 0, 42.5, or 85 ppm arsenite from gestation day 8 to 18. After birth, groups (n = 25) of offspring received topical 12-O-tetradecanoyl phorbol-13-acetate (TPA) (2 microg twice a week) for 36 weeks and were killed; nonskin tumors were assessed. Arsenic increased adrenal cortical adenomas (ACAs; 25%-29%) compared with control (0%) independent of TPA in all male groups. Arsenic increased urinary bladder (UB) hyperplasia in males, but only with TPA. Arsenic induced ACAs in all female groups (control 0%; arsenic 17%-26%). Arsenic-treated females had UB hyperplasia in most groups (control 0%; arsenic 26%-32%), with 2 UB papillomas. All arsenic-treated females had uterine hyperplasia (26%-40%; control 4%) independent of TPA, and 3 had uterine tumors. Thus, arsenic in utero rapidly induces ACAs and uterine and UB preneoplasias in Tg.AC mice, showing transplacental carcinogenic potential in yet another strain of mice.

Metallothionein protection of cadmium toxicity.

The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-molecular weight, cysteine-rich protein called metallothionein (MT) in Cd toxicology. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an association between human and rodent Cd exposure and prostate cancers, especially in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicology, with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is critical for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.

Arsenic-induced malignant transformation of human keratinocytes: involvement of Nrf2.

Arsenic is a well-known human skin carcinogen but the underlying mechanisms of carcinogenesis are unclear. Transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism, and emerging data suggest that constitutive activation of Nrf2 contributes to malignant phenotype. In the present study when an immortalized, nontumorigenic human keratinocyte cell line (HaCaT) was continuously exposed to an environmentally relevant level of inorganic arsenite (100 nM) for 28 weeks, malignant transformation occurred as evidenced by the formation of highly aggressive squamous cell carcinoma after inoculation into nude mice. To investigate the mechanisms involved, a broad array of biomarkers for transformation were assessed in these arsenic-transformed cells (termed As-TM). In addition to increased secretion of matrix metalloproteinase-9 (MMP-9), a set of markers for squamous differentiation and skin keratinization, including keratin-1, keratin-10, involucrin, and loricrin, were significantly elevated in As-TM cells. Furthermore, As-TM cells showed increased intracellular glutathione and elevated expression of Nrf2 and its target genes, as well as generalized apoptotic resistance. In contrast to increased basal Nrf2 activity in As-TM cells, a diminished Nrf2-mediated antioxidant response induced by acute exposure to high doses of arsenite or tert-butyl hydroxyquinone occurred. The findings that multiple biomarkers for malignant transformation observed in As-TM cells, including MMP-9 and cytokeratins, are potentially regulated by Nrf2 suggest that constitutive Nrf2 activation may be involved in arsenic carcinogenesis of skin. The weakened Nrf2 activation in response to oxidative stressors observed in As-TM cells, coupled with acquired apoptotic resistance, would potentially have increased the likelihood of transmittable oxidative DNA damage and fixation of mutational/DNA damage events.

Marked liver tumorigenesis by Helicobacter hepaticus requires perinatal exposure.

BACKGROUND: Although severe hepatitis and liver tumors occur in a high percentage of A/J male mice naturally infected with Helicobacter hepaticus, these effects have not been observed after injection of adult mice with the bacteria. OBJECTIVES: We tested the hypothesis that perinatal exposure to the bacteria is required for liver tumorigenesis. METHODS: A/J female mice were infected by intragastric (ig) or intraperitoneal (ip) treatment with 1.5 x 10(8) H. hepaticus before pregnancy. We examined offspring at progressive time intervals, including some kept until natural death in old age. A/J, BALB/c, and C57BL/6 weanling male mice were similarly treated ig with the bacteria and observed for up to 2 years. RESULTS: After ip bacterial infection of A/J females, 41% of their male offspring developed hepatitis and 33% had hepatocellular tumors, including 18% with hepatocellular carcinoma. Treatment by the ig route resulted in a similar incidence of hepatitis in offspring (35%) but fewer total liver tumors (8%) and carcinomas (4%). By contrast, ig instillation of H. hepaticus in weanling A/J, C57BL/6, or BALB/c mice resulted in low incidence of hepatitis (0-20%) and few liver tumors, despite presence of bacteria confirmed in feces. CONCLUSIONS: Results indicate that a high incidence of liver tumors in mice infected with H. hepaticus requires perinatal exposure. Contributing perinatal factors could include known high sensitivity of neonatal liver to tumor initiation, and/or modulation of immune response to the bacterium or its toxins. Mechanisms of human perinatal sensitivity to such phenomena can be studied with this model.

Urogenital carcinogenesis in female CD1 mice induced by in utero arsenic exposure is exacerbated by postnatal diethylstilbestrol treatment.

Transplacental inorganic arsenic carcinogenicity, together with postnatal exposure to diethylstilbestrol or tamoxifen, was studied. Pregnant CD1 mice received 85 ppm arsenic in the drinking water from gestation days 8 to 18 and were allowed to give birth. Groups (n = 35) of female offspring were injected s.c. on postpartum days 1 through 5 with diethylstilbestrol (2 microg/pup/d) or tamoxifen (10 microg/pup/d) and observed for 90 weeks. Arsenic alone induced some urogenital system tumors, including mostly benign tumors of the ovary and uterus, and adrenal adenoma. Diethylstilbestrol alone induced some tumors (primarily cervical) but when given after in utero arsenic, it greatly enhanced urogenital tumor incidence, multiplicity, and progression. For instance, compared with the incidence of urogenital malignancies in the control (0%), arsenic alone (9%), and diethylstilbestrol alone (21%) groups, arsenic plus diethylstilbestrol acted synergistically, inducing a 48% incidence of malignant urogenital tumors. Of the urogenital tumors induced by arsenic plus diethylstilbestrol, 80% were malignant, and 55% were multiple site. Arsenic plus diethylstilbestrol increased ovarian, uterine, and vaginal tumors, and urinary bladder proliferative lesions, including three transitional cell carcinomas. Tamoxifen alone did not increase urogenital tumors or affect arsenic-induced neoplasia but did increase arsenic-induced uroepithelial proliferative lesions. Uterine and bladder carcinoma induced by arsenic plus diethylstilbestrol greatly overexpressed estrogen receptor-alpha (ER-alpha) and pS2, an estrogen-regulated gene. In neonatal uteri, prenatal arsenic increased ER-alpha expression and enhanced estrogen-related gene expression induced by postnatal diethylstilbestrol. Thus, arsenic acts with estrogens to enhance production of female mouse urogenital cancers.

Fetal onset of aberrant gene expression relevant to pulmonary carcinogenesis in lung adenocarcinoma development induced by in utero arsenic exposure.

Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-alpha (ER-alpha) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-beta-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. In utero arsenic exposure also induced overexpression of alpha-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-alpha expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-alpha expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-alpha activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood.

Aberrant DNA methylation and gene expression in livers of newborn mice transplacentally exposed to a hepatocarcinogenic dose of inorganic arsenic.

Our prior work showed that brief exposure of pregnant C3H mice to inorganic arsenic-induced hepatocellular carcinoma (HCC) formation in adult male offspring. The current study examined the early hepatic events associated with this oncogenic transformation. Pregnant mice were exposed to a known carcinogenic dose of arsenic (85 ppm) in the drinking water from gestation days 8 to 18. The dams were allowed to give birth and liver samples from newborn males were analyzed for arsenic content, global DNA methylation and aberrant expression of genes relevant to the carcinogenic process. Arsenic content in newborn liver reached 57 ng/g wet weight, indicating arsenic had crossed the placenta, reached the fetal liver and that significant amounts remained after birth. Global methylation status of hepatic DNA was not altered by arsenic in the newborn. However, a significant reduction in methylation occurred globally in GC-rich regions. Microarray and real-time RT-PCR analysis showed that arsenic exposure enhanced expression of genes encoding for glutathione production and caused aberrant expression of genes related to insulin growth factor signaling pathways and cytochrome P450 enzymes. Other expression alterations observed in the arsenic-treated male mouse newborn liver included the overexpression of cdk-inhibitors and stress response genes including increased expression of metallothionein-1 and decreased expression of betaine-homocysteine methyltransferase and thioether S-methyltransferase. Thus, transplacental exposure to arsenic at a hepatocarcinogenic dose induces alterations in DNA methylation and a complex set of aberrant gene expressions in the newborn liver, a target of arsenic carcinogenesis.

Global gene expression associated with hepatocarcinogenesis in adult male mice induced by in utero arsenic exposure.

Our previous work has shown that exposure to inorganic arsenic in utero produces hepatocellular carcinoma (HCC) in adult male mice. To explore further the molecular mechanisms of transplacental arsenic hepatocarcinogenesis, we conducted a second arsenic transplacental carcinogenesis study and used a genomewide microarray to profile arsenic-induced aberrant gene expression more extensively. Briefly, pregnant C3H mice were given drinking water containing 85 ppm arsenic as sodium arsenite or unaltered water from days 8 to 18 of gestation. The incidence of HCC in adult male offspring was increased 4-fold and tumor multiplicity 3-fold after transplacental arsenic exposure. Samples of normal liver and liver tumors were taken at autopsy for genomic analysis. Arsenic exposure in utero resulted in significant alterations (p < 0.001) in the expression of 2,010 genes in arsenic-exposed liver samples and in the expression of 2,540 genes in arsenic-induced HCC. Ingenuity Pathway Analysis revealed that significant alterations in gene expression occurred in a number of biological networks, and Myc plays a critical role in one of the primary networks. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis of selected genes/proteins showed > 90% concordance. Arsenic-altered gene expression included activation of oncogenes and HCC biomarkers, and increased expression of cell proliferation-related genes, stress proteins, and insulin-like growth factors and genes involved in cell-cell communications. Liver feminization was evidenced by increased expression of estrogen-linked genes and altered expression of genes that encode gender-related metabolic enzymes. These novel findings are in agreement with the biology and histology of arsenic-induced HCC, thereby indicating that multiple genetic events are associated with transplacental arsenic hepatocarcinogenesis.

Arsenicals in maternal and fetal mouse tissues after gestational exposure to arsenite.

Exposure of pregnant C3H/HeNCR mice to 42.5- or 85-ppm of arsenic as sodium arsenite in drinking water between days 8 and 18 of gestation markedly increases tumor incidence in their offspring. In the work reported here, distribution of inorganic arsenic and its metabolites, methyl arsenic and dimethyl arsenic, were determined in maternal and fetal tissues collected on gestational day 18 of these exposure regimens. Tissues were collected from three females and from associated fetuses exposed to each dosage level. Concentrations of total speciated arsenic (sum of inorganic, methyl, and dimethyl arsenic) were higher in maternal tissues than in placenta and fetal tissues; total speciated arsenic concentration in placenta exceeded those in fetal tissues. Significant dosage-dependent (42.5 ppm versus 85 ppm of arsenite in drinking water) differences were found in total speciated arsenic concentrations in maternal lung (p<0.01) and liver (p<0.001). Total speciated arsenic concentrations did not differ significantly between dosage levels for maternal blood or for fetal lung, liver, and blood, or for placenta. Percentages of inorganic, methyl, or dimethyl arsenic in maternal or fetal tissues were not dosage-dependent. Over the range of total speciated arsenic concentrations in most maternal and fetal tissues, dimethyl arsenic was the most abundant arsenical. However, in maternal liver at the highest total speciated arsenic concentration, inorganic arsenic was the most abundant arsenical, suggesting that a high tissue burden of arsenic affected formation or retention of methylated species in this organ. Tissue concentration-dependent processes could affect kinetics of transfer of inorganic arsenic or its metabolites from mother to fetus.

Inorganic arsenite-induced malignant transformation of human prostate epithelial cells.

Although several epidemiologic studies show an association between arsenic exposure and prostate cancer, it is still unknown whether human prostate epithelial cells are directly susceptible to arsenic-induced transformation. This study was designed to determine whether the nontumorigenic human prostate epithelial cell line RWPE-1 could be malignantly transformed in vitro by arsenite. RWPE-1 cells were continuously exposed to 5 micro M arsenite and monitored for signs of transformation, assessed as changes in matrix metalloproteinase-9 levels. After 29 weeks of exposure, the arsenite-exposed RWPE-1 cells (referred to as CAsE-PE) showed a marked increase in matrix metalloproteinase-9 secretion, a common finding in prostate malignancies. Malignant transformation was confirmed when CAsE-PE cells produced aggressive undifferentiated malignant epithelial tumors in nude mice. The tumors stained positive for human prostate-specific antigen, confirming their origin. These results are the first report of arsenite-induced malignant transformation of a human epithelial cell line and provide an important in vitro model for studying the mechanisms underlying arsenic-induced carcinogenesis in humans.

Metallothionein-I/II double knockout mice are hypersensitive to lead-induced kidney carcinogenesis: role of inclusion body formation.

Lead is an environmental nephrotoxicant and probable human carcinogen. Elucidating factors predisposing populations to lead toxicity is an important public health issue. Recently, we found that metallothionein-I/-II double knockout (metallothionein-null) mice that are unable to produce the major forms of metallothionein do not produce lead inclusion bodies, which are thought to mitigate lead toxicity, and were sensitive to the subchronic toxic effects of lead exposure (10 weeks), showing modestly diminished renal function and nephromegaly compared with wild-type (WT) mice. It is unclear how this knockout might impact lead carcinogenesis. Thus, the effects of lead(II) acetate were tested in groups (n = 25) of male metallothionein-null and WT mice receiving drinking water with 0, 1,000, 2,000, or 4,000 parts per million lead for up to 104 weeks. Renal proliferative lesions (adenoma and cystic tubular atypical hyperplasia) were much more common and more severe in lead-exposed metallothionein-null mice than in WT mice. A metastatic renal cell carcinoma also occurred in a lead-treated metallothionein-null mouse, whereas none occurred in WT mice. Lead-induced renal proliferative lesions showed marked overexpression of cyclin D1, a common feature of human renal tumors. Renal lead-containing nuclear inclusion bodies were frequently observed in WT mice but did not form in metallothionein-null mice. Metallothionein was often found associated with the outer portion of these inclusion bodies. Thus, the metallothionein-null mice cannot form renal inclusion bodies, even after protracted lead exposure, and this increases the carcinogenic potential of lead. Poor production of metallothionein may predispose human populations to lead carcinogenicity.

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide donor of the diazeniumdiolate class with potent antineoplastic activity.

We have previously shown that nitric oxide (NO) inhibits growth and induces differentiation and apoptosis in acute myeloid leukemia cells, with the HL-60 human myeloid leukemia line being particularly sensitive to NO-mediated cytolysis. With the goal of identifying a prodrug that can target NO to the leukemia cells without inducing NO-mediated systemic hypotension, we have screened a series of O(2)-aryl diazeniumdiolates designed to be stable at physiological pH but to release NO upon reaction with glutathione. O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) proved to be the most active antiproliferative agent among those tested in HL-60 cells, with an IC(50) of 0.2-0.5 microM. After 5 days of exposure to 0.5 micro M JS-K, HL-60 cells had differentiated and acquired some of the phenotypic features of normal monocytes. One- to 2-day treatment with JS-K at concentrations of 0.5-1 microM resulted in apoptosis induction in a concentration- and caspase-dependent manner. JS-K also inhibited the growth of solid tumor cell lines but to a lesser extent than HL-60 cells. JS-K was administered i.v. to nonobese diabetic-severe combined immune deficient mice at doses of up to 4 micromol/kg without inducing significant hypotension. The growth of s.c. implanted HL-60 cells was reduced by approximately 50% when the mice received i.v. injections three times/week with 4 micromol/kg boluses of JS-K. Histological examination of tumor explants from JS-K-treated animals revealed extensive necrosis. Similar results were seen with s.c. human prostate cancer (PPC-1) xenografts. Our data indicate that JS-K is a promising lead compound for the possible development of a novel class of antineoplastic agents.

Down-regulation of von Hippel-Lindau protein in N-nitroso compound-induced rat non-clear cell renal tumors.

Non-clear cell rat kidney tumors, inducible by N-nitroso compounds but lacking mutations in the von Hippel--Lindau (VHL) coding sequence, were examined for other VHL alterations. Neither mutations nor DNA methylation was detected in a putative promoter region. By immunohistochemistry, however, VHL protein level was evidently reduced in six of the eight eosinophilic renal epithelial tumors and in all the ten nephroblastomas. Immunoblotting of normal kidney detected two VHL proteins of 20 and 22kDa in a 16-day-old fetal rat but only 20kDa protein in an adult rat. This is the first demonstration of VHL alteration at the protein level.

Treatment with lovastatin, cholestyramine or niacin alters K-ras membrane association in mouse lung in a strain-dependent manner: results in females.

Hypocholesterolemic drugs may themselves increase (cholestyramine, CS) or decrease (lovastatin, Lov) peripheral tissue de novo cholesterol biosynthesis. This will alter the abundance of prenyl groups and potentially increase (CS) or decrease (Lov) K-ras membrane localization, with possible pro- or anti-carcinogenic effects (K-ras is a proto-oncogene frequently mutated in lung cancer). Female A/J, Swiss, and C57BL/6 mice were fed 2 or 4% CS, 1% niacin, or injected with Lov three (Lov-3x) or five (Lov-5x) times per week. After three weeks, serum cholesterol and triglycerides were determined enzymatically. Total, membrane, and cytoplasmic K-ras proteins were determined in lung homogenates by immunoprecipitation followed by Western blotting with a K-ras specific antibody. CS feeding increased membrane K-ras as hypothesized in A/J and C57BL/6 mice, but had no effect in Swiss mice. Lov failed in all three strains to reduce membrane K-ras, and resulted in an increase in total K-ras in A/J and C57BL/6 mice, while again lacking effect in Swiss mice. Niacin had no effect on K-ras protein in any mouse strain. These results differ from our published results for male mice of the same strains, particularly for A/J mice. Increased amounts of K-ras protein in the membrane fraction of A/J females (but not males) treated with either Lov or CS imply that if K-ras were to become mutated, CS could result in increased lung tumorigenesis and Lov would be less likely to be protective in females. In the light of these data, both sexes should be included in future animal and human chemoprevention trials.

Alterations in membrane-bound and cytoplasmic K-ras protein levels in mouse lung induced by treatment with lovastatin, cholestyramine, or niacin: effects are highly mouse strain dependent.

Agents that either increase (cholestyramine, CS) or decrease (lovastatin, Lov) de novo peripheral cholesterol synthesis may increase (CS) or decrease (Lov) ras protein membrane localization by altering protein prenylation, and potentially have pro- or anti-carcinogenic effects. Male A/J, Swiss, and C57/BL6 mice were treated with 2 or 4% CS, 1% dietary niacin, or 25mg/kg of Lov three times per week (Lov-3X) or five times per week (Lov-5X). After 3 weeks, serum cholesterol and triglycerides were determined enzymatically. Membrane and cytoplasmic K-ras proteins in lung were determined by immunoprecipitation followed by western blotting with a K-ras specific antibody. Results confirmed the hypothesis only in isolated instances. A/J mice had a significant 30% increase in cytoplasmic K-ras and a 40% decrease in membrane K-ras from Lov treatment, as predicted. C57/BL6 mice had a significant 77% increase in membrane K-ras, as expected from CS feeding. At variance with the hypothesis, Swiss mice had increased levels (3-28%) of membrane K-ras with all treatments (including Lov), and C57/BL6 mice treated with Lov had a 58-78% increase in cytoplasmic K-ras without any reduction in the levels of membrane K-ras. Niacin, predicted to have no effect on ras membrane localization, decreased cytoplasmic K-ras in A/J mice, increased both membrane and cytoplasmic K-ras in Swiss mice, and had no effect in C57/BL6 mice. Results may have differed from those predicted because of strain-dependent differences in response to the cholesterol-lowering agents. A difference in response among the mouse strains suggests that individual genetic differences may alter the effect of hypocholesterolemic agents on K-ras membrane localization, and potentially the risk of ras-dependent cancer.

Toxicogenomic analysis of aberrant gene expression in liver tumors and nontumorous livers of adult mice exposed in utero to inorganic arsenic.

Arsenic is a known human carcinogen. We have reported that brief exposure of pregnant C3H mice to arsenite in their drinking water during gestation induced hepatocellular carcinoma (HCC) in male offspring after they became adults. Tumor formation is typically associated with multiple gene expression changes, and this study examined aberrant gene expression associated with transplacental arsenic hepatocarcinogenesis. Liver tumors and nontumorous liver samples were taken at necropsy from adult male mice exposed in utero to either 42.5 or 85 ppm arsenic as sodium arsenite or unaltered water from day 8 to 18 of gestation. Total RNA was extracted and subjected to microarray analysis. Among 600 genes, arsenic-induced HCC showed a higher rate of aberrant gene expression (>2-fold and p < 0.05, 14%) than spontaneous tumors (7.8%). Overexpression of alpha-fetoprotein, c-myc, cyclin D1, proliferation-associated protein PAG, and cytokeratin-18 were more dramatic in arsenic-induced HCC than spontaneous tumors. In nontumorous liver samples of arsenic-exposed animals, 60 genes (10%) were differentially expressed, including the increased expression of alpha-fetoprotein, c-myc, insulin-like growth factor binding protein-1, superoxide dismutase, glutathione S-transferases, and CYP2A4, and the depressed expression of CYP7B1. Real-time RT-PCR analysis largely confirmed these findings. This toxicogenomic analysis revealed several aberrant gene expression changes associated with transplacental arsenic carcinogenesis. It is indeed remarkable that expression changes occurred in adulthood even though arsenic exposure ended during gestation. Some of these aberrantly expressed genes could play a role in the development of arsenic-induced tumors, at least in the liver.

Mechanisms underlying arsenic carcinogenesis: hypersensitivity of mice exposed to inorganic arsenic during gestation.

Inorganic arsenic is an important human carcinogen of unknown etiology. Defining carcinogenic mechanisms is critical to assessing the human health hazard of arsenic exposure but requires appropriate model systems. It has proven difficult to induced tumors in animals with inorganic arsenic alone. Several groups have studied the carcinogenic potential of inorganic arsenic in rodents, finding it to act as co-promoter or co-carcinogen, but not as a complete carcinogen. As gestation is a time of high sensitivity to chemical carcinogenesis, we performed two in utero exposure studies with inorganic arsenic. In the first study, pregnant mice received drinking water containing sodium arsenite at 0 (control), 42.5 and 85 ppm arsenic from gestation day 8 to 18, and the offspring were observed for up to 90 weeks. As adults, male offspring developed hepatocellular carcinoma (HCC) and adrenal tumors after in utero arsenite exposure. Although liver tumors were not induced by arsenic in female offspring, they did develop lung carcinoma, ovarian tumors, and uterine and oviduct preneoplasia. In a second study, the same doses of arsenic were used and the skin tumor promoting phorbol ester, TPA, was applied to the skin after birth in an effort to promote skin tumors potentially initiated by arsenic in utero. TPA did not promote dermal tumors after in utero arsenite exposure. Otherwise, results from the second chronic study largely duplicated the first and, irrespective of additional TPA exposure, arsenic exposure in utero induced HCC and adrenal tumors in males and ovarian tumors in females. In addition, combined arsenic and TPA induced a significant increase in hepatocellular tumors in female offspring, although arsenic alone was not effective. Thus, in utero inorganic arsenic exposure can act as a complete carcinogen in mice, with brief exposures consistently inducing tumors at several sites. In addition, it appears gestational arsenic can act as a tumor initiator in the female mouse liver, inducing liver lesions that can be promoted by TPA.