Complete Carcinogen Research Articles

05/02444 Economic and policy implications of public support for ethanol production in california's San Joaquin Valley

During petroleum refining, a large number of products are generated which have varying chemical and physical properties. These are known in the industry as petroleum streams. In order to characterize their carcinogenic activity, a number of these commercially produced streams were administered to C3H/HeJ mice in chronic dermal bioassays. The bioassays were conducted using one of two study designs: the first set of test materials was applied for a lifetime and the second set for 24 months. In the lifetime study, the last mice in the test groups survived for periods of 31 to 32 months. Middle distillates, boiling in the range 115–390°C, were found to decrease the lifespan of exposed mice compared to controls or streams of higher and lower boiling ranges. These middle distillate streams included straight run kerosine, hydrodesulfurized middle distillate, straight run middle distillate, light catalytic cracked distillate, and 90/10% and 70/30% mixtures of the last two. The middle distillate streams also proved to be active as carcinogens, with tumor incidence ranging from 16 to 67%. Light alkylate naphtha, heavy catalytic reformed naphtha, vacuum residuum, and unleaded gasoline did not demonstrate significant carcinogenic potency. Heavy thermal cracked naphtha, heavy catalytic cracked naphtha, and hydrotreated light naphthenic distillate were dermal carcinogens of low potency in this study. Administration of light catalytic cracked naphtha led to a low incidence of very late developing tumors with a mean latency of 118 weeks. Application of the 0.1% solution of catalytic cracked clarified oil in toluene did not result in a significant incidence of tumors, but the 10% solution caused almost 100% mortality and 100% tumor incidence in 12 months. There was no correlation between carcinogenic potency and the indices of irritation, alopecia, erythema, and scabbing. Only two of the streams tested, hydrotreated light naphthenic distillate and 10% catalytic cracked clarified oil, contain polynuclear aromatic hydrocarbons (PNAs) and may be presumed to be complete carcinogens. The middle distillates and heavy naphthas are nonmutagenic and essentially free of PNAs. Their activity may result from promotion of already-initiated skin sites. Where comparisons could be made, reducing the exposure period from a lifetime (29–32 months) to 24 months did not change the evaluations of stream carcinogenicity except in the case of light catalytic cracked naphtha where six of the seven mice that developed tumors did so after 24 months.

Epigenetic Effectiveness of Complete Carcinogens: Specific Interactions of Polycyclic Aromatic Hydrocarbons and Aminoazo Dyes with Cholesterol and Apolipoprotein A-I

During a co-precipitation of cholesterol (Chol) and slight amounts of polycyclic aromatic hydrocarbons (PAHs) or aminoazo dyes (AZOs) in aqueous albumin solution, complex particles are formed; on their surfaces apolipoproteins with an amphipathic alpha-helix (e.g. apoA-I) are more or less firmly adsorbed. An efficacy index can be calculated from the strength of the hydrophobic interactions between apoA-I and the [Chol/PAH]- or [Chol/AZO]-complex, and the solubility of the PAH or AZO in an aqueous medium, which correlates to the carcinogenicity of these compounds. A short-term test for PAHs and AZOs is described, in which the efficacy index can be determined in the simplest manner without any great expenditure on equipment. The previous results suggest that the parent compounds of the PAHs and AZOs can be involved in a specific interaction with cholesterol-domains of the plasma membrane of a cell. The changes in membrane fluidity and architecture caused by these specific interactions could modulate the distribution and/or activity of membrane proteins which are critical to the regulation of cellular proliferation.

Deletion of Bid Impedes Cell Proliferation and Hepatic Carcinogenesis

Mechanisms that control the proliferation capability of the initiated cells during hepatocarcinogenesis are still largely unclear. We investigated the role of a pro-death Bcl-2 family protein, Bid, in liver tumor development using a neonatal diethylnitrosamine model. Diethylnitrosamine was administrated to 15-day-old wild-type and bid-null mice. The development of microfoci at the early stage and of gross tumors at the later stage was compared between the two groups of mice. Both microfoci and gross tumor development were significantly retarded in the bid-null mice, despite reduced cell death as measured by TUNEL staining. Further studies indicated that there were significantly less proliferating cells in diethylnitrosamine-treated bid-null livers. The regulation of cell proliferation by Bid was confirmed in two other systems not involving carcinogenesis. Hepatocyte proliferation following partial hepatectomy and T lymphocyte proliferation following anti-CD3 stimulation were both retarded in bid-null mice. Thus, these studies revealed a previously undisclosed function of Bid in regulating cell proliferation, which can be important to tumor development. Furthermore, the role of Bid in promoting hepatocarcinogenesis is in contrast to its reported role in suppressing myeloid leukemia and thus suggests an organ- and/or etiology-specific role of the Bcl-2 family proteins in regulating oncogenesis.

Silibinin inhibits ultraviolet B radiation-induced mitogenic and survival signaling, and associated biological responses in SKH-1 mouse skin

Ultraviolet B (UVB) radiation is a complete skin carcinogen causing DNA damage as a tumor-initiating event and activating signaling cascades that play a critical role in its tumor-promoting potential. Recently we reported that a naturally occurring flavonoid, silibinin, protects UVB-induced skin damages and prevents photocarcinogenesis. Here we examined silibinin efficacy on acute and chronic UVB-caused mitogen-activated protein kinases (MAPKs) and AKT activation and associated biological responses in SKH-1 hairless mouse skin. A single UVB exposure at 180 mJ/cm2 dose resulted in varying degrees of ERK1/2, JNK1/2, MAPK/p38 and AKT phosphorylation at various time-points in mouse skin; however, topical application of silibinin prior to or immediately after UVB exposure, or its dietary feeding strongly inhibited the activation of these molecules at all the time-points examined. Stronger effects of silibinin towards inhibition of UVB-caused phosphorylation of MAPKs and AKT were also observed in a chronic UVB (180 mJ/cm2/day for 5 days) exposure protocol. Immunohistochemical analysis of chronically exposed skin sections showed that silibinin treatment in all three protocols increases UVB-induced p53-positive cells and decreases UVB-caused cell proliferation, apoptotic and sunburn cells. These findings suggest that silibinin inhibits UVB-induced MAPK and AKT signaling and increases p53 in mouse skin, and that these effects of silibinin possibly lead to a decrease in UVB-caused proliferation and apoptosis, which might, in part, be responsible for its overall efficacy against photocarcinogenesis.

Ultraviolet‐free phototherapy

Ultraviolet (UV) radiation phototherapy has been used for decades in the management of common skin diseases. On the other hand, UV radiation is a complete carcinogen and as a consequence, UV phototherapy is usually not used for the long-term management of children and young adults and in combination with topical or systemic immunosuppressants. The therapeutic effectiveness of a new UV-free irradiation device in the treatment of patients with atopic hand and/or foot eczema was studied. In a single-blinded trial 10 patients with atopic hand and/or foot eczema were treated with a sham irradiation device and with the new developed UV-free irradiation device DermoDyne during the following 4 weeks. All irradiation's lasted 30 min per treatment three times weekly. UV-free irradiation was found to induce a significant clinical improvement of atopic hand and foot eczema (P = 0.0001) in marked contrast to the sham-irradiation (P = 0.39). Our studies demonstrate that visible light can be successfully used for the treatment of patients with atopic eczema.

Balsamodendron mukul suppresses benzoyl peroxide and ultraviolet light induced tumor promotional events in Swiss mice

The current study evaluates the efficacy of Balsamodendron mukul Hook ex. Stocks, an indigenous plant, in the modulation of benzoyl peroxide (BPO) treated and ultraviolet (UV) light irradiated tumor promotional events. BPO is a well-known tumor promoter while UV radiations are capable of acting as complete carcinogens. Treatment of the dorsal portions of the mice skins with BPO (20 mg/0.2 ml/animal) and subsequent UV irradiation (0.420 J/m 2/s) caused a significant depletion of reduced glutathione (GSH), it’s metabolizing and phase II enzymes ( p < 0.01). Also, down regulation of the activities of antioxidant enzymes and up regulation of ornithine decarboxylase activity and enhancement in the synthesis of DNA, malondialdehyde (MDA) and hydrogen peroxide (H 2O 2) was observed. However, topical pretreatment with the extract of B. mukul not only restored the content of GSH, MDA and H 2O 2 but it also recovered the activities of above-mentioned enzymes ( p < 0.05) and prevented synthesis of DNA ( p < 0.05) significantly. Although the lower dose was not very effective, the higher dose showed promising results against oxidative stress and hyperproliferative response of BPO and UVB radiations. From the present data we conclude that B. mukul is potentially strong enough to modulate the events of skin tumor promotion.

Protein kinase C epsilon is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas.

Chronic exposure to UV radiation (UVR), especially in the UVA (315-400 nm) and UVB (280-315 nm) spectrum of sunlight, is the major risk factor for the development of nonmelanoma skin cancer. UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. We found that protein kinase C epsilon (PKCepsilon), a member of the phospholipid-dependent threonine/serine kinase family, is an endogenous photosensitizer, the overexpression of which in the epidermis increases the susceptibility of mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. The PKCepsilon transgenic mouse (FVB/N) lines 224 and 215 overexpressed 8- and 18-fold PKCepsilon protein, respectively, over endogenous levels in basal epidermal cells. UVR exposure (1 kJ/m(2) three times weekly) induced irreparable skin damage in high PKCepsilon-overexpressing mouse line 215. However, the PKCepsilon transgenic mouse line 224, when exposed to UVR (2 kJ/m(2) three times weekly), exhibited minimum cutaneous damage but increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks. UVR exposure of PKCepsilon transgenic mice compared with wild-type littermates (1) elevated the levels of neither cyclobutane pyrimidine dimer nor pyrimidine (6-4) pyrimidone dimer, (2) reduced the appearance of sunburn cells, (3) induced extensive hyperplasia and increased the levels of mouse skin tumor promoter marker ornithine decarboxylase, and (4) elevated the levels of tumor necrosis factor alpha (TNFalpha) and other growth stimulatory cytokines, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. The role of TNFalpha in UVR-induced cutaneous damage was evaluated using PKCepsilon transgenic mice deficient in TNFalpha. UVR treatment three times weekly for 13 weeks at 2 kJ/m(2) induced severe cutaneous damage in PKCepsilon transgenic mice (line 215), which was partially prevented in PKCepsilon-transgenic TNFalpha-knockout mice. Taken together, the results indicate that PKCepsilon signals UVR-induced TNFalpha release that is linked, at least in part, to the photosensitivity of PKCepsilon transgenic mice.

A protective role for matrix metalloproteinase-3 in squamous cell carcinoma.

Elevated expression of matrix metalloproteinase-3 (MMP-3/stromelysin-1) is associated with a variety of tumor types, although its in vivo functional role remains unclear. In human and murine squamous cell carcinoma (SCC), MMP-3 is expressed in the stromal compartment at all of the stages of tumor progression and is expressed by the malignant epithelial cells in late-stage, highly invasive tumors. To elucidate whether MMP-3 plays a causal role during SCC, wild-type and MMP-3 null mice were subjected to chemical carcinogenesis procedures by topical application of either the complete carcinogen 1-methyl-3-nitro-1-nitroso-guanidine or two-stage initiation and promotion with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. Contrasting with our expectations, tumors originating on MMP-3 null mice had enhanced initial tumor growth rates as compared with control animals, although there was no difference in tumor onset or incidence. This elevated rate in growth was coupled with an elevated proliferative index and a reduced vasculature density but with no significant effect on apoptosis. Tumors from MMP-3 null mice had a prevalence of undifferentiated spindle tumors as compared with controls, which was concomitant with a higher percentage of MMP-3 null mice evidencing surface lung metastases. Tumor progression in MMP-3 null mice was inversely associated with leukocyte infiltration, in which an overall reduction in tumor-associated macrophages and neutrophils was evident. We propose that MMP-3 is expressed as a protective response and plays an important role in host defense during SCC tumorigenesis.

Suppressive effects of dietary curcumin on the increased activity of renal ornithine decarboxylase in mice treated with a renal carcinogen, ferric nitrilotriacetate

Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to act as a biological response modifier in various disorders. We have reported previously that the dietary supplementation of curcumin enhances the activities of antioxidant and phase II metabolizing enzymes in mice (M. Iqbal, S.D. Sharma, Y. Okazaki, M. Fujisawa, S. Okada, Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY mice: possible role in protection against chemical carcinogenesis and toxicity, Pharmacol and Toxicol. 92 (2003) 33_38.) and inhibits ferric nitrilotriacetate (Fe-NTA) induced oxidative injury of lipids and DNA in vitro (M. Iqbal, Y. Okazaki, S. Okada, In vitro curcumin modulates Ferric Nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H(2)O(2))-induced peroxidation of microsomal membrane lipids and DNA damage, Teratogenesis Carcinogenesis and Mutagenesis Supplement 23 (2003) 151-160.). In our present study, Fe-NTA, a known complete renal carcinogen, which generate ROS in vivo, was given intraperitoneally to mice and curcumin was tested for its ability to inhibits oxidative stress and the activity of ornithine decarboxylase (ODC) as well as histopathological changes in the kidney. Substantial changes in glutathione, antioxidant enzymes as well as changes in phase II metabolizing enzymes were observed in the kidney at 12 h after treatment with Fe-NTA (9.0 mg Fe/kg body weight). Effect of oxidative stress induced by Fe-NTA were also demonstrated by the increase in lipid peroxidation as monitored by formation of thiobarbituric acid-reactive substances and 4-hydroxy-2-nonenal (HNE)-modified proteins in kidney. Likewise, the level of protein carbonyl contents, an indicator of protein oxidation was also increased after Fe-NTA administration. However, the changes in these parameters were restored to normal in curcumin-pretreated mice. The ODC activity in the kidney was significantly increased by Fe-NTA, while the increased ODC activity induced by Fe-NTA was normalized in curcumin-pretreated mice. In addition, curcumin pretreatment almost completely prevented kidney biomolecules from oxidative damage and protected the tissue against observed histopathological alterations.

Altered apoptotic response in MCF 10A cells treated with the equine estrogen metabolite, 4-hydroxyequilenin

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues including the breast. 4-Hydroxyequilenin (4-OHEN), a major catechol metabolite of equine estrogens present in Premarin ®, an estrogen replacement formulation, has been shown to induce apoptosis and DNA damage in human breast cancer cells. It also has the potential to be a tumor initiator or promoter and complete carcinogen. To further understand the effects and mechanisms of equine catechol estrogen metabolite 4-OHEN action in vitro, human non-tumorigenic mammary epithelial MCF 10A cell line was used to study the toxic effects of 4-OHEN. In this study, we observed that 4-OHEN caused dose-dependent increases in apoptosis and DNA damage as measured by the DAPI nuclear screening assay and the Comet assay, respectively. Interestingly, cells treated with 100 nM 4-OHEN biweekly for 4 weeks became resistant to cisplatin-induced apoptosis. The resistance to apoptosis of the 100 nM 4-OHEN-treated cells was through multiple regulatory mechanisms. Compared to the DMSO-treated cells, the 100 nM 4-OHEN-treated cells had higher GSH levels and total SOD activity, and a stronger GSH response after cisplatin treatment. Expression levels of several genes involved in cell growth, DNA repair, and apoptosis were either up- or down-regulated. These data indicate that long-term low-level equine estrogen metabolite exposure could induce DNA damage and initiate cells to become resistant to apoptosis.

Deficiency of NRH:quinone oxidoreductase 2 increases susceptibility to 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene-induced skin carcinogenesis.

NRH:Quinone oxidoreductase 2 (NQO2) is an enzyme that catalyzes the reductive metabolism of quinones. C57BL/6 NQO2-/- mice lacking NQO2 gene expression were generated in our laboratory. The dorsal skin of NQO2-deficient mice was exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene alone (complete carcinogen) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (initiation/promotion model) to determine the in vivo role of NQO2 in chemical carcinogenesis. The NQO2-/- mice showed significantly increased tumor frequency with DMBA + TPA when compared with their wild-type littermates. The benzo(a)pyrene + TPA also showed increase in tumor incidence in NQO2-/- mice but to a less extent than DMBA. DMBA alone resulted in low frequency of tumor development with no difference in susceptibility between wild-type and NQO2-/- mice. Benzo(a)pyrene alone failed to induce tumors in either wild-type or NQO2-/- mice. Histologic analysis of the NQO2-/- mice tumors demonstrated proliferative activity. The treatment of NQO2-/- mice skin with benzo(a)pyrene failed to significantly increase tumor suppressor protein p53 and p53-regulated growth-related protein p21 and proapoptotic protein Bax as observed in case of wild-type mice. These results demonstrate that NQO2 protects against DMBA- and benzo(a)pyrene-induced skin carcinogenesis and suggest that NQO2 protection might be against tumor promotion. The results also suggest that lack of induction of p53, p21, and Bax proteins might contribute to increased sensitivity of NQO2-/- mice skin to benzo(a)pyrene carcinogenicity.

Oral and dermal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cutaneous papillomas and squamous cell carcinomas in female hemizygous Tg.AC transgenic mice.

Tg.AC mice develop epidermal papillomas in response to treatment with dermally applied nongenotoxic and complete carcinogens. The persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a multi-site rodent carcinogen and tumor promoter that induces the formation of papillomas in Tg.AC mice. To examine the dose-response relationship and compare dermal and oral routes of exposure for TCDD-induced skin papillomas, female Tg.AC mice were exposed dermally to average daily doses of 0, 2.1, 7.3, 15, 33, 52, 71, 152, and 326 ng TCDD/kg/day or 0, 75, 321, and 893 ng TCDD/kg body weight by gavage for 26 weeks. The incidence of cutaneous papillomas was increased in a dose-dependent manner, and tumors developed earlier with higher exposure to TCDD regardless of route of administration. Increased incidences of cutaneous squamous cell carcinomas were observed in mice exposed to dermal (> or =52 ng/kg) and oral (893 ng/kg) TCDD. Higher gavage doses than dermal exposure doses were required to induce papillomas and squamous cell carcinomas. Despite a linear correlation between administered dose and terminal skin concentrations, the incidence of tumor formation was lower in the gavage study than in the dermal study with respect to mean terminal skin TCDD concentrations. These studies demonstrate that, although Tg.AC mice are less responsive to TCDD by gavage than by dermal exposure, the induction of skin neoplasms is a response to systemic exposure and not solely a local response at the site of dermal application. Differences in response between the routes of exposure may reflect pharmacokinetic differences in the delivery of TCDD to the skin over the duration of the study.

Targeted over-expression of mPGES-1 and elevated PGE2 production is not sufficient for lung tumorigenesis in mice.

There is a significant body of evidence suggesting that enzymes involved in arachidonic acid metabolism and their eicosanoid products play a role in various cancers, having both pro- and antitumorigenic effects. The goal of this study was to further define the role microsomal prostaglandin E synthases (mPGES-1) play in lung tumorigenesis. Transgenic mice were created with targeted over-expression of human mPGES-1 in the alveolar and airway epithelial cells using an SP-C promoter driven construct. Transgene positive (mPGES-1+) mice were shown to significantly over-express functional mPGES-1 in the lung and more specifically in alveolar type II cells. To study the effects of mPGES-1 over-expression in lung tumor formation, mice were exposed to a complete carcinogen protocol with a single injection of urethane or an initiation/promotion model with a single injection of 3-methylcholanthrene (MCA) followed by multiple injections of butylated hydroxytoluene (BHT). mPGES-1+ mice did not show a significant difference in tumor multiplicity or tumor size at 10, 16, 19 or 30 weeks after urethane injection compared with mPGES-1- mice. No significant difference was seen in tumor incidence, multiplicity or size at 19 weeks after treatment with MCA/BHT. Western blots verified that mPGES-1 expression was increased in tumors versus uninvolved tissue of both mPGES-1+ and mPGES-1- mice with overall expression being significantly higher in mPGES-1+ mice. Cyclooxygenase-2 levels were elevated in tumors in both groups. From these studies we conclude that over-expression of mPGES-1 and highly elevated PGE2 production are not sufficient to induce lung tumors.

Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-kappaB in mouse epidermal cl41 cells.

Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs.

PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells.

5-Methylchrysene has been found to be a complete carcinogen in laboratory animals. However, the tumor promotion effects of (±)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) remain unclear. In the present work, we found that 5-MCDE induced marked activator protein-1 (AP-1) activation in Cl41 cells. 5-MCDE also induced a marked activation of phosphatidylinositol 3-kinase (PI-3K). Inhibition of PI-3K impaired 5-MCDE–induced AP-1 transactivation, suggesting that PI-3K is an upstream kinase involved in AP-1 activation by 5-MCDE. Furthermore, we found that Akt is a PI-3K downstream mediator for 5-MCDE–induced AP-1 transactivation, whereas another PI-3K downstream kinase, p70S6K, was not involved in AP-1 activation by 5-MCDE. Moreover, inhibition of Akt activation blocked 5-MCDE–induced activation of extracellular signal–regulated protein kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs), whereas it did not affect p38K activation. Consistently, overexpression of a dominant-negative mutant of ERK2 or JNK1 blocked the AP-1 activation by 5-MCDE. These results demonstrate that 5-MCDE is able to induce AP-1 activation, and the AP-1 induction is specifically through a PI-3K/Akt–dependent and p70S6K-independent pathway.

Experimental cancer studies of chlorinated by-products.

Chlorinated drinking water contains a number of different by-products formed during the chlorination process from organic matter. The carcinogenicity of only a fraction of them have been evaluated in experimental animals. The focus has been on compounds and groups of compounds that are most abundant in chlorinated drinking water or the in vitro toxicity data have suggested genotoxic potential. From trihalomethanes, chloroform causes liver tumors in mice and female rats and renal tumors in male mice and rats. Tumor formation by chloroform is strongly associated with cytotoxicity and regenerative cell proliferation in tissues and that has been considered to be one determinant of its carcinogenicity. From halogenic acetic acids, dichloroacetic acid (DCA) and trichlotoacetic acid (TCA) are hepatocarcinogenic in mice and DCA in male rats. Their genotoxicity is equivocal and nongenotoxic mechanisms, such as peroxisome proliferation and hypomethylation of DNA in the liver, likely contribute to tumor development. From chlorinated furanones (CHFs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a multisite carcinogen in rats (e.g. in thyroid glands and liver) and it has caused DNA damage in vivo. MX may be a complete carcinogen because it also has promoter properties in vitro. Chlorinated drinking water may also contain brominated by-products providing the raw water contains bromide. At least some of them (bromodichloromethane, bromoform) have been shown to be carcinogenic in laboratory animals. Altogether, although several by-products have been shown to have carcinogenic potential in laboratory animals, it not yet possible to state which compounds or groups of by-products cause the cancer risk in chlorinated drinking water. The cellular mechanisms of their effects and these effects at low concentrations are still poorly understood. The few studies with mixtures of these by-products suggest that the mixture effects may be complex and unpredictable (inhibitory, additive, synergistic).

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.

Safety assessment of dichlorophene and chlorophene.

Dichlorophene is a halogenated phenolic compound that functions as a bacteriocide and fungicide in cosmetics. Chlorophene is a halogenated phenolic compound that functions as a biocide and preservative in cosmetics. Dichlorophene was reported to be used in a total of five cosmetic formulations at concentrations of 0% to 1.0%, but Chlorophene was not reported to be used. Dichlorophene is prohibited for use in cosmetic ingredients in Japan. In Europe, the maximum authorized concentration allowed for Dichlorophene is 0.5% and for Chlorophene is 0.2%. The major impurity of Dichlorophene is the trimer 4-chloro-2,6-bis(5-chloro-2-hydroxybenzyl)phenol. In rats, Dichlorophene sulfate, Dichlorophene monoglucuronide, and Dichlorophene diglucuronide were the major metabolites of Dichlorophene and were excreted, mainly in the urine. The glucuronic acid conjugate, the sulfate ester conjugate, and two minor metabolites of Chlorophene were the metabolites found in rat urine. Chlorophene was incompletely absorbed through the rat skin. These chemicals exhibited low toxicity in acute oral toxicity studies in several animal species. Some evidence of toxicity with both chemicals was found in short-term oral toxicity studies in mice and rats; nephropathy was the principal finding. Chronic toxicity data were not available for Dichlorophene. Rats and mice dosed with Chlorophene for 2 years had a dose-related and sex-related increase in the severity of nephropathy. In animal tests, Dichlorophene and Chlorophene were ocular irritants. No inhalation toxicity data were available for these ingredients. Dichlorophene up to 10% concentration resulted in no to minimal irritation when applied to the intact and abraded skin of rabbits. Chlorophene was severely irritating to rabbits in most dermal irritation studies. Studies on guinea pigs gave positive and negative results in sensitization tests of Dichlorophene. A dose-related contact hypersensitivity response to Chlorophene was reported in mice. No reproductive or developmental toxicity data were available for Dichlorophene, but there was some evidence of non-dose-dependent developmental toxicity with Chlorophene in rabbits. Dichlorophene was positive in the Ames mutagenicity assay, but not in mammalian or fruit fly test systems. Chlorophene was mutagenic in four in vitro mammalian test systems. Carcinogenicity studies for Dichlorophene were not found. Neoplasms were not observed in rats treated with Chlorophene for 2 years; however a significant incidence of neoplasms was observed in mice so treated. A 1-year National Toxicology Program (NTP) study concluded that Chlorophene was a cutaneous irritant and a weak skin tumor promoter but had no activity as an initiator or complete carcinogen. Dichlorophene was not a sensitizer in clinical dermal sensitization tests. Some reactions to Chlorophene occurred in some, but not all, clinical dermal sensitization tests. Positive photopatch tests to Dichlorophene were found in 13/469 patients. Although these ingredients were ocular irritants at high concentrations, the risk at concentrations which are actually used in cosmetic formulations was uncertain. Overall, the available data were insufficient to support safety of Dichlorophene or Chlorophene. Additional data needed include (1) method of manufacture and impurities data (especially the trimer in Dichlorophene); (2) photosensitization and photocarcinogenicity data for Dichlorophene; (3) dermal reproductive and developmental toxicity data for Dichlorophene (as a function of dose); and (4) ocular irritation at concentration of use, if available.

Estrogen receptor-mediated regulation of oxidative stress and DNA damage in breast cancer.

Estrogens have been implicated to be complete carcinogens in breast and gynecologic tissues. Possible mechanisms may include differential metabolism with subsequent formation of reactive oxygen species and/or a receptor-mediated pathway, which may also involve indirect modulation of intracellular redox state. Estrogen-mediated oxidative DNA damage in mammary gland epithelia includes the induction of 8-oxo-2'-deoxyguanosine, both in vitro and in vivo, thereby suggesting a role for oxidative stress in the initiation and/or progression of breast neoplasia. In order to study this phenomenon, we have treated estrogen receptor alpha (ER-alpha)-positive MCF-7 cells and ER-alpha-negative MDA-MB-231 cells with 10 nM 17beta-estradiol (E2), while measuring changes in antioxidant status and sensitivity to DNA damage by peroxide. Treatment of MCF-7 cells with E2 resulted in a marked decrease in the ability for these cells to metabolize peroxide, which paralleled a decrease in catalase activity and total glutathione levels. These observations also correlated with an increased sensitivity to peroxide-induced DNA damage. The estrogen-induced effects were all opposed by the anti-estrogen tamoxifen. In addition, the estrogen-mediated down regulation of peroxide metabolism, catalase activity, and sensitivity to DNA damage were not observed in the MDA-MB-231 cell line. Treatment of MCF-7 cells with E2 also resulted in increased glutathione peroxidase, superoxide dismutases (I) and (II) and glucose-6-phosphate dehydrogenase activities. Therefore, in this breast cancer model antioxidant status is modulated through the actions of the ER. The data may explain some of the estrogen-induced pro-oxidant effects previously reported in vivo. In addition, this is the first report indicating that E2 is capable of inducing an increase in sensitivity to oxidative DNA damage through an ER-mediated mechanism.

Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers

Arsenic is a recognized human carcinogen and development of rodent models remains a critically important research objective. Since gestation can be a period of high sensitivity to chemical carcinogenesis, we have performed a series of transplacental carcinogenicity studies in mice with inorganic arsenic. In this study, groups of pregnant C3H mice received drinking water containing sodium arsenite (NaAsO2) at 0, 42.5 and 85 p.p.m. arsenic ad libitum from days 8 to 18 of gestation. These doses of arsenic were well tolerated. Dams delivered normally and at weaning (4 weeks) offspring were randomly put into groups (n = 25) of males or females according to maternal dose. In an attempt to promote skin cancers initiated by transplacental arsenic, duplicate groups of control or arsenic exposed offspring were topically exposed to 12-O-tetradecanoyl phorbol-13-acetate (TPA; 2 micro g/0.1 ml acetone, twice/week) from 4 to 25 weeks of age. Irrespective of TPA exposure, male offspring showed arsenic-induced dose-related increases in hepatocellular carcinoma incidence and multiplicity, as well as increases in adrenal tumor incidence and multiplicity. In female offspring, an increase in epithelial ovarian tumors occurred with arsenic exposure regardless of TPA exposure. Females also showed pre-neoplastic lesions of the reproductive tract, including hyperplasia of the uterus and oviduct, after arsenic but independent of TPA exposure. Although TPA had no effect on skin tumors, it promoted arsenic initiated liver tumors in females and lung tumors in both sexes. Thus, inorganic arsenic, as a single agent, can consistently act as a complete transplacental carcinogen in mice, inducing tumors at multiple sites, and as a tumor initiator in some tissues. Skin tumors were not initiated by arsenic in mouse fetuses possibly indicating tissue-specific mechanisms of action. This study indicates that gestation is a period of high sensitivity to arsenic carcinogenesis.