Zymbal Gland Research Articles

Multi-organ carcinogenicity by inhalation exposure to 2-Bromopropane in rats.

The purpose of this study was to investigate the carcinogenicity of 2-bromopropane (2-BP) in rats. Male and female F344 rats were exposed by whole body inhalation to 2-BP vapor at concentrations of 0, 67, 200, and 600 ppm for 6h/day, 5 days/week for 2 years. All rats of both sexes exposed to 600 ppm died or became moribund within 85 weeks. Death/moribunditywas caused by 2-BP induced tumors. In males, significantly increased tumors were malignant Zymbal's gland tumors; sebaceous adenoma and basal cell carcinoma of the skin/appendage; adenocarcinoma of the small/large intestine; follicular cell adenoma of the thyroid; fibroma of the subcutis, and malignant lymphoma of the lymph node. In addition, an increased trend in tumor incidence was found in the preputial gland, lung, forestomach, pancreas islet, brain, and spleen. In females, significantly increased tumors were adenocarcinoma and fibroadenoma of the mammary gland, squamous cell papilloma of the vagina, and large granular lymphocytic leukemia of the spleen. In addition, an increased trend in tumor incidence was found in Zymbal's gland, the clitoral gland, skin, large intestine, pancreas islet, uterus, and subcutis. Particularly, malignant Zymbal's gland tumors were induced even in males exposed to the lowest concentration, 67 ppm. Two-year inhalation exposure to 2-BP resulted in multi-organ carcinogenicity in rats. Based on sufficient evidence of carcinogenicity in this study, 2-BP has the potential to be a human carcinogen.

The EDA-deficient mouse has Zymbal's gland hypoplasia and acute otitis externa.

ABSTRACTIn mice, rats, dogs and humans, the growth and function of sebaceous glands and eyelid Meibomian glands depend on the ectodysplasin signalling pathway. Mutation of genes encoding the ligand EDA, its transmembrane receptor EDAR and the intracellular signal transducer EDARADD leads to hypohidrotic ectodermal dysplasia, characterised by impaired development of teeth and hair, as well as cutaneous glands. The rodent ear canal has a large auditory sebaceous gland, the Zymbal’s gland, the function of which in the health of the ear canal has not been determined. We report that EDA-deficient mice, EDAR-deficient mice and EDARADD-deficient rats have Zymbal’s gland hypoplasia. EdaTa mice have 25% prevalence of otitis externa at postnatal day 21 and treatment with agonist anti-EDAR antibodies rescues Zymbal’s glands. The aetiopathogenesis of otitis externa involves infection with Gram-positive cocci, and dosing pregnant and lactating EdaTa females and pups with enrofloxacin reduces the prevalence of otitis externa. We infer that the deficit of sebum is the principal factor in predisposition to bacterial infection, and the EdaTa mouse is a potentially useful microbial challenge model for human acute otitis externa.

2,2’-dichloro-4,4’-methylenedianiline – inhalable fraction and vapours. Documentation of proposed values of occupational exposure limits (OELs)

2,2’-dichloro-4,4’-methylenedianiline – inhalable fraction and vapours. Documentation of proposed values of occupational exposure limits (OELs)

3,3'-Dimethylbenzidine and its salts – inhalable fraction. Documentation of proposed values of occupational exposure limits (OELs)

3,3'-Dimethylbenzidine (3,3'-DMB, DMB, o-tolidine) is a solid used (as water-soluble dihydrochloride salt (dimethylbenzidine · 2HCl)) in the production of azopigments, polyurethane elastomers and plastics for coating. Small amounts are also used in diagnostic tests in laboratories. Occupational exposure to dimethylbenzydine occurs mainly during the production and use of pigments to dye textiles, plastics, paper and leather. In 2005–2014, dimethylbenzidine was used in Poland in 18–30 workplaces, where 135–280 people each year (mainly women) were exposed. No epidemiological data and information related to toxic effects of DMB in humans was found in the available literature. The LD50 value (median lethal dose) after single intragastric administration of 3,3'-dimethylbenzidine to rats was 404 mg/kg. After repeated exposure of laboratory animals, liver, kidney, thyroid injury and hematological changes were noted. In the Ames tests with metabolic activation, it was found that metabolites of 3,3'-dimethylbenzidine show stronger mutagenic action than the parent compound. 3,3'-DMB induced also chromosome aberrations and exchange of sister chromatids in in vitro tests. Although 3,3'-dimethylbenzidine is a derivative of carcinogenic benzidine, carcinogenic effects on humans have not been proven. However, research provides data about carcinogenic effect of 3,3'-DMB in animals. After subcutaneous administration of 3,3'-dimethylbenzidine and its dihydrochloride salt in drinking water, Zymbal's and mammary glands tumors, and cancers of uterus, skin, liver, hematopoietic system, small and large intestine were observed in rats. IARC classified 3,3'-dimethylbenzidine in the 2B group (a supposed carcinogenic agent for humans), whereas ACGIH – in the A3 group (proved carcinogenic effect on animals and unknown carcinogenic effect for humans). The European Union (according to the CLP classification) has listed 3,3'-DMB in the 1B category with the inscription "H350 – can cause cancer". The permissible concentrations for 3,3'-dimethylbenzidine have been established in some European countries only (Austria, Slovenia and Switzerland) as 0.03 mg/m3. The basis of the proposed maximum concentration value (MAC-TWA) for 3,3'-dimethylbenzidine and its salts was a risk assessment of cancer in male rats chronically exposed to 3,3'-dimethylbenzidine dihydrochloride in drinking water. Taking into account the cancer risk at the level of 10-4, a concentration of 0.03 mg/m3 for the MAC-TWA value was proposed. There are no basis to determine the short-term value (STEL) and biological limit values (BLV). It was also proposed to label the compound with "Carc 1B", which indicates that it is a carcinogen category 1B, and "skin" – the absorption of substances through the skin may be as important as an inhalation route.

Assessment of DNA Binding and Oxidative DNA Damage by Acrylonitrile in Two Rat Target Tissues of Carcinogenicity: Implications for the Mechanism of Action.

Exposure to acrylonitrile induces formation of tumors at multiple sites in rats, with females being more sensitive. The present study assessed possible mechanisms of acrylonitrile tumorigenicity, covalent DNA binding, DNA breakage, and oxidative DNA damage, in two target tissues, the brain and Zymbal's glands, of sensitive female Fischer (F344) and Sprague-Dawley (SD) rats. One group received acrylonitrile in drinking water at 100 ppm for 28 days. Two other groups were administered either acrylonitrile in drinking water at 100 ppm or drinking water alone for 27 days, followed by a single oral gavage dose of 11 mg/kg bw 14C-acrylonitrile on day 28. A positive control group received a single dose of 5 mg/kg bw of 7-14C-benzo[a]pyrene, on day 27 following the administration of drinking water for 26 days. Using liquid scintillation counting, no association of radiolabeled acrylonitrile with brain DNA was found. In accelerator mass spectrometry analysis, the association of 14C of acrylonitrile with DNA in brains was detected and was similar in both strains, which may reflect acrylonitrile binding to protein as well as to DNA. Nucleotide 32P-postlabeling assay analysis of brain samples from rats of both strains yielded no evidence of acrylonitrile DNA adducts. Negative conventional comet assay results indicate the absence of direct DNA strand breaks in the brain and Zymbal's gland in both strains of rats dosed with acrylonitrile. In both rat strains, positive results in an enhanced comet assay were found only in brain samples digested with formamidopyrimidine-DNA glycosylase but not with human 8-hydroxyguanine-DNA glycosylase, indicating possible oxidative DNA damage, other than 8-oxodG formation. In conclusion, definitive evidence of DNA binding of acrylonitrile in the brain and Zymbal's gland was not obtained under the test conditions. A role for oxidative stress in tumorigenesis in the brain but not Zymbal's gland may exist.

A miRNA signature for an environmental heterocyclic amine defined by a multi-organ carcinogenicity bioassay in the rat.

Heterocyclic amines (HCAs) produced during high-temperature cooking have been studied extensively in terms of their genotoxic/genetic effects, but recent work has implicated epigenetic mechanisms involving non-coding RNAs. Colon tumors induced in the rat by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have altered microRNA (miRNA) signatures linked to dysregulated pluripotency factors, such as c-Myc and Krüppel-like factor 4 (KLF4). We tested the hypothesis that dysregulated miRNAs from PhIP-induced colon tumors would provide a "PhIP signature" for use in other target organs obtained from a 1-year carcinogenicity bioassay in the rat. Downstream targets that were corroborated in the rat were then investigated in human cancer datasets. The results confirmed that multiple let-7 family members were downregulated in PhIP-induced skin, colon, lung, small intestine, and Zymbal's gland tumors, and were associated with c-myc and Hmga2 upregulation. PhIP signature miRNAs with the profile mir-21high/mir-126low/mir-29clow/mir-215low/mir-145low were linked to reduced Klf4 levels in rat tumors, and in human pan-cancer and colorectal cancer. It remains to be determined whether this PhIP signature has predictive value, given that more than 20 different genotoxic HCAs are present in the human diet, plus other agents that likely induce or repress many of the same miRNAs. Future studies should define more precisely the miRNA signatures of other HCAs, and their possible value for human risk assessment.

Zymbal Gland (Auditory Sebaceous Gland) Carcinoma Presenting as Otitis Externa in a Pet Rat (Rattus norvegicus)

A 1-year-old sexually intact male fancy rat (Rattus norvegicus) was presented for unilateral purulent otitis externa and weight loss. A small amount of bloody discharge was noted at the ostium of the left ear canal. The canal and the surrounding tissue were firm to the touch. Mild left-sided enophthalmos and facial nerve paralysis were noted. Otoscopic examination demonstrated caseous debris, and a swab of the external ear canal demonstrated a mixed population of rods and cocci on cytological evaluation. Bacterial culture of the external ear canal grew Escherichia coli. The rat was initially placed on oral and topical antimicrobial therapy. Despite treatment, the condition of the affected ear deteriorated and the rat continued to lose weight. Other clinical signs observed at this time included a worsening of the facial nerve paralysis, head tilt, and circling. A biopsy sample of the swollen tissue surrounding the left ear canal was collected; however, the rat died the day following the biopsy procedure. Histopathology of the biopsied tissue revealed an invasive neoplasm consistent with a Zymbal gland (auditory sebaceous gland) carcinoma. Zymbal glands are modified sebaceous glands surrounding the ear canal of rodents. Although chemically induced neoplasia of these glands is frequently observed, spontaneous tumor formation is much less common. Tumors of the Zymbal gland should be considered as underlying causes for unilateral otitis externa in rats.

Bacterial Prostatitis Enhances 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine (PhIP)-Induced Cancer at Multiple Sites.

Dietary carcinogens, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and chronic inflammation have each been implicated as etiologic agents in prostate cancer. We hypothesized that bacterial prostatitis would accelerate PhIP-induced preinvasive lesions in the rat prostate. Male Fischer 344 rats were assigned into 4 groups: Control (untreated), PhIP (200 ppm in the diet for 20 weeks), Escherichia coli (E. coli, prostatic inoculation in week 10), or PhIP + E. coli. Study animals were monitored for a total of 52 weeks and were euthanized as necessary based on strict criteria for health status and tumor burden. Animals treated with E. coli initially developed acute and chronic inflammation in all lobes of the prostate, whereas inflammation was observed predominantly in the ventral lobe at time of death. PhIP + E. coli-treated animals exhibited a marked decrease in survival compared with PhIP-alone-treated animals as a result of an increase in the number of invasive cancers that developed at multiple sites, including the skin, small intestine, and Zymbal's gland. Despite their earlier mortality, PhIP + E. coli-treated animals developed an increased average number of precancerous lesions within the prostate compared with PhIP-treated animals, with a significantly increased Ki-67 index. Multiplexed serum cytokine analysis indicated an increase in the level of circulating IL6 and IL12 in PhIP + E. coli-treated animals. Elevated serum IL6 levels correlated with the development of precancerous lesions within the prostate. These results suggest that bacterial infections and dietary carcinogens, two conceivably preventable cancer risk factors, may synergistically promote tumorigenesis.

A case of zymbal gland carcinoma complicated with leukemia in Wistar rat

A one year old breeder male Wistar rat showed hard mass of 2x5x3 cm size on the right external ear canal and right side of the head. On cut surface, it comprised mainly of a yellowish friable material with occasional cystic spaces. Liver and spleen were enlarged. Microscopically, the neoplasm comprised polygonal cells arranged in islands and trabeculae supported by a fine to moderate fibrovascular stroma. Neoplastic islands and trabeculae form a peripheral layer of flattened to cuboidal epithelial cells with scanty amounts of eosinophilic, granular cytoplasm; round to oval nuclei with finely stippled chromatin and up to 4 distinct nucleoli (basaloid reserve cells); and a central area containing polygonal cells with abundant microvacuolated cytoplasm and round nuclei (sebaceous differentiation). Nuclei were round with finely stippled chromatin and one to two nucleoli. On microscopic examination, liver revealed centrilobular hepatocellular degeneration and infiltration of tumor cells in the sinusoidal and periportal areas while examination of spleen showed diffuse infiltration of round to oval neoplastic cells. It was diagnosed as a rare case of zymbal gland carcinoma complicated with leukemic changes in liver and spleen.

Abstract 594: Effect of caloric restriction and high fat diet on azoxymethane-induced carcinogenesis in LETO and OLETF rats

Abstract We showed recently that diabetic and mildly obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats are more susceptible to azoxymethane (AOM)-induced Zymbal gland and intestinal carcinogenesis than control Long-Evans Tokushima Otsuka (LETO) rats. In the present study, we examined the effect of caloric restriction (CR) and high-fat diet (HFD) on AOM-induced carcinogenesis in these strains. Experiment 1: Six-week-old male LETO and OLETF rats (n=21) were given s.c. injections of 15 mg/kg AOM once weekly for 3 weeks, then fed, Group 1) control diet, Group 2) 20% CR diet, or Group 3) HFD (control diet with 10% safflower oil), and killed at 36 weeks of age. In LETO rats, the incidences of colon tumors (adenoma plus adenocarcinoma) in Groups 1-3 were 45%, 33% and 55%, respectively. Average number of tumors/rat in CR group was fewer than that in HFD group (P<0.05). In OLETF rats, the incidences of colon tumors in Groups 1-3 were 76%, 48% and 62%, respectively. Zymbal gland tumors in CR group developed later than those in control and HFD groups in OLETF rats. Experiment 2: Male LETO and OLETF rats (n=5) were fed control diet, 20% CR diet or HFD for 5 weeks, and killed at 24 weeks of age. In LETO rats, serum triglyceride, total cholesterol, insulin and leptin levels decreased by 20% CR, and increased by HFD compared to the control group. In OLETF rats, those levels decreased by 20% CR, and increased by HFD except leptin. Expression of hepatic IGF-1 and Sirt-1 mRNA elevated by 20% CR in both strains. The liver of OLETF rat HFD group showed severe fatty change histologically. In conclusion, 20% CR inhibited, and HFD promoted colon carcinogenesis in LETO rats. In OLETF rats, 20% CR inhibited, but HFD did not promote colon carcinogenesis because of early development of Zymbal gland tumors. Hyperinsulinemia and/or hyperlipidemia may exert the promoting effect in rat carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 594. doi:1538-7445.AM2012-594

Inhibition or Enhancement by 4 Pacific Island Food Plants Against Cancers Induced by 2 Amino–3-Methylimidazo[4,5-f]Quinoline in Male Fischer 344 Rats

A 1-yr carcinogenicity bioassay was conducted in rats fed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), simultaneously with AIN-76/ high-fat (HF) diet alone, or with 10% starch replaced with kumara, pineapple, coconut, or taro, prepared as for a human diet. All of the non-IQ treated control, kumara, pineapple, or taro but not coconut-fed rats survived to 1 yr. None of the IQ-fed animals survived to 1 yr and although there were minor survival time differences among the groups, none was statistically significant. At sacrifice, IQ/HF controls had tumors in the skin, Zymbal's gland, ear canal, oral cavity, liver, and small intestine, totaling 32 among 20 animals. Kumara-fed rats had a similar tumor distribution but no tumors in the ear or oral cavity, and a total of 27 tumors among 20 animals, whereas pineapple-fed rats showed a somewhat lower tumor incidence (23/20 animals), including no small intestine lesions. Unexpectedly, a higher tumor incidence, especially of skin tumors, was seen in coconut and taro-fed animals (35/20 and 41/20 animals, respectively). In particular, the incidence of malignant liver tumors and gastrointestinal tumors were significantly increased in the taro-fed group in comparison with the kumara group.

High Susceptibility to Zymbal Gland and Intestinal Carcinogenesis in Diabetic Otsuka Long-Evans Tokushima Fatty Rats

Diabetes mellitus (DM) and obesity are believed to be risk factors for colorectal cancer in humans. In experiment 1, male nondiabetic Long-Evans Tokushima Otsuka (LETO) rats and Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model animal of type 2 DM, were whole-body X-irradiated (4 Gy) at 6 and 8 weeks of age and euthanized at 78 weeks of age (n=15, respectively). The incidences of small intestine adenocarcinoma in LETO and OLETF rats were 0% and 30%, respectively. In experiment 2, male LETO and OLETF rats (n=24, respectively) were given s.c. injections of 15 mg/kg azoxymethane (AOM) once weekly for 3 weeks and euthanized at 36 weeks of age. The incidences of Zymbal gland tumors in LETO and OLETF rats were 0% and 67%, respectively (P<0.001), whereas those of small intestine adenocarcinoma were 0% and 43% (P<0.001) and those of cecum/colon adenocarcinoma were 46% and 79% (P<0.05), respectively. Fatty change of hepatocytes was common in OLETF rats (63%) but not in LETO rats. Serum triglyceride and free fatty acid levels in OLETF rats were significantly higher than in LETO rats at sacrifice, whereas serum insulin levels in OLETF rats were very diverse. These data suggest that hyperlipidemia plays a significant role in high susceptibility to lower intestinal tract carcinogenesis in OLETF rats; this strain is susceptible to AOM-induced Zymbal gland carcinogenesis.

Application of the Margin of Exposure (MoE) approach to substances in food that are genotoxic and carcinogenic: Example: Benzene, CAS: 71–43-2

The role of genotoxic events in the aetiology of benzene induced tumours cannot be ruled out. Dose response modelling of the data for benzene gave a BMDL10 for female Zymbal gland carcinoma of 17.6 mg/kg-bw/d following adjustment to daily average doses. The MOEs ranged from 2 x 10 6 to 0.4 x 10 6 depending on the assumptions used in the exposure estimation.

Two-year inhalation study of carcinogenicity and chronic toxicity of 1,4-dioxane in male rats

Carcinogenicity and chronic toxicity of 1,4-dioxane were examined by inhalation exposure of 50 male F344 rats to 1,4-dioxane vapor at 0 (clean air), 50, 250, or 1250 ppm (v/v) for 6 h/day, 5 days/wk, and 104 wk. Survival rates of 250 and 1250 ppm-exposed groups were decreased near the end of the 2-yr exposure period, due probably to the occurrence of malignant tumors. A statistically significant but marginal decrement of terminal body weight (<10%) was found in the 1250 ppm-exposed group, suggesting slight systemic toxicity. Significant changes in plasma levels of AST, ALT, ALP, and γ-GTP and relative weight of the liver occurred in the 1250 ppm-exposed group. Dose-dependent and statistically significant increases in incidences of nasal squamous cell carcinomas, hepatocellular adenomas, and peritoneal mesotheliomas were found primarily in the 1250 ppm-exposed group. The incidences of renal cell carcinomas, fibroadenomas in the mammary gland, and adenomas in the Zymbal gland were also increased dose-dependently. Preneoplastic lesions occurred in the nasal cavity and liver of the 1,4-dioxane-exposed groups. As nonneoplastic lesions, the significantly increased incidences of nuclear enlargement, atrophy, and respiratory metaplasia in the nasal cavity were noted at 50 ppm and above. A LOAEL (lowest observed adverse effect level) was determined at 50 ppm for the nasal endpoint of general chronic toxicity. This study provides clear evidence of carcinogenicity for 1,4-dioxane in male rats. A cytotoxic-proliferative and in vivo genotoxic mode of action is suggested to operate in 1,4-dioxane-induced carcinogenesis.

Protective versus promotional effects of white tea and caffeine on PhIP-induced tumorigenesis and β-catenin expression in the rat

A 1 year carcinogenicity bioassay was conducted in rats treated with three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)/high-fat (HF) diet, followed by 2% white tea (wt/vol), 0.05% epigallocatechin-3-gallate (EGCG) or 0.065% caffeine as sole source of fluid intake. Thirty-two percent of the PhIP/HF controls survived to 1 year, compared with 50, 48.7 and 18.2% in groups given white tea, EGCG and caffeine, respectively. After 1 year, PhIP/HF controls had tumors in the colon, skin, small intestine, Zymbal's gland, salivary gland and pancreas. For all sites combined, excluding the colon, tumor incidence data were as follows: PhIP/HF 69.5%, PhIP/HF + EGCG 48.7%, PhIP/HF + white tea 46.9% and PhIP/HF + caffeine 13.3%. Unexpectedly, a higher incidence of colon tumors was detected in rats post-treated with white tea (69%) and caffeine (73%) compared with the 42% incidence in PhIP/HF controls. In the colon tumors, beta-catenin mutations were detected at a higher frequency after caffeine posttreatment, and there was a shift toward more tumors harboring substitutions of Gly34 with correspondingly high protein and messenger RNA expression seen for both beta-catenin and c-Myc. c-Myc expression exhibited concordance with tumor promotion, and there was a concomitant increase in cell proliferation versus apoptosis in colonic crypts. A prior report described suppression of PhIP-induced colonic aberrant crypts by the same test agents, but did not incorporate a HF diet. These findings are discussed in the context of epidemiological data which do not support an adverse effect of tea and coffee on colon tumor outcome-indeed, some such studies suggest a protective role for caffeinated beverages.

Benzene-induced Cancers: Abridged History and Occupational Health Impact

Benzene-induced cancer in humans was first reported in the late 1920s. Carcinogenesis findings in animals were not reported conclusively until 1979. Industry exploited this "discrepancy" to discredit the use of animal bioassays as surrogates for human exposure experience. The cardinal reason for the delay between first recognizing leukemia in humans and sought-after neoplasia in animals centers on poor design and conduct of experimental studies. The first evidence of carcinogenicity in animals manifested as malignant tumors of the zymbal glands (sebaceous glands in the ear canal) of rats, and industry attempted to discount this as being irrelevant to humans, as this organ is vestigial and not present per se in humans. Nonetheless, shortly thereafter benzene was shown to be carcinogenic to multiple organ sites in both sexes of multiple strains and multiple species of laboratory animals exposed via various routes. This paper presents a condensed history of the benzene bioassay story with mention of benzene-associated human cancers.

Hydroquinone: An Evaluation of the Human Risks from its Carcinogenic and Mutagenic Properties

The toxicology of hydroquinone has been reviewed on a number of previous occasions. This review targets its potential for carcinogenicity and possible modes of carcinogenic action. The evaluation made by IARC (1999) of its carcinogenic risk to humans was that hydroquinone is not classifiable as to its carcinogenicity to humans (Group 3). This evaluation was based on inadequate evidence in humans and limited evidence in experimental animals. The epidemiological information comes from four cohort studies involving occupational exposures. A cohort of lithographers, some of whom had worked with hydroquinone, had an excess of malignant melanoma based on five cases, but only two of the cases had reported exposure to hydroquinone. In a study of photographic processors the number of exposed individuals was uncertain and the numbers of cases of individual cancer sites were small. In view of the statistical power limitations of these studies for individual diagnostic categories of cancers, they are not considered to be informative with regard to the carcinogenicity of hydroquinone. A cohort of workers with definite and lengthy exposure to hydroquinone, during either its manufacture or its use, had low cancer rates compared with two comparison populations; the reason for the lower than expected rates is unclear. In a motion picture film processing cohort there were significant excess malignancies of the respiratory system among workers engaged in developing, where there was exposure to hydroquinone as well as other chemicals. There was no information on tobacco smoking habits and no dose-response relationship. Hydroquinone has been shown reproducibly to induce benign neoplasms in the kidneys of male F344 rats dosed orally either by gavage (25 and 50 mg/kg body weight) or diet (0.8%). The gavage study has been evaluated in considerable detail. This evaluation showed that all renal tubule adenomas and all cases of renal tubule atypical hyperplasia occurred in areas of severe or end-stage chronic progressive nephropathy and that the neoplasms were not otherwise confined to any particular part of the kidney. It is likely that the mode of carcinogenic action of hydroquinone is exacerbation of this natural disease process. Hydroquinone is mutagenic in vitro and in vivo, having caused genotoxicity or chromosomal aberrations in rodent bone-marrow cells. At least a portion, if not all, of the chromosomal effects are caused by interference by hydroquinone or its metabolites with chromosomal segregation, probably due to interaction with mitotic spindle proteins. However, the dose routes used to demonstrate these effects in almost all of the studies in vivo were intraperitoneal or subcutaneous injection, which were considered inappropriate. There were five studies by the oral route. These included a mouse bone-marrow cell micronucleus test in which a weak, marginally positive response was obtained following a single oral dose of 80 mg/kg body weight. The remaining oral route studies all showed no significant effect. They included a mouse bone-marrow cell micronucleus test in which there was no genotoxic activity after exposure to a diet containing 0.8% hydroquinone for 6 days; two 32P-post-labeling assays, one with targets of Zymbal gland, liver, and spleen in Sprague-Dawley rats, the other with the kidney as target in F344 rats; and the last oral assay was for 8-hydroxydeoxyguanosine adducts in F344 rat kidney DNA. Thus, the evidence (and the database) for any genotoxic effect in vivo is sparse and none has been observed in kidney. While glutathione conjugates could be responsible for the tumor induction, careful histology seems to show that the most actively toxic of several glutathione compounds tested, 2,3,5-triglutathion-S-yl hydroquinone, targets a very specific region of the kidney, the outer stripe of the outer medulla (OSOM), whereas hydroquinone-associated adenomas are more randomly distributed and occur in the cortex as well as the medulla. A nongenotoxic mode of action that involves exacerbation of a spontaneously occurring rodent renal disease, chronic progressive nephropathy (CPN), is proposed and evaluated. This disease is particularly prominent in male rats and the evidence is consistent with an absence of any human counterpart; therefore, the increased incidence of renal tubule adenomas in hydroquinone-dosed male rats is without human consequence.

Enhancing effects of simultaneous treatment with sodium nitrite on 2-amino-3-methylimidazo[4,5-f]quinoline-induced rat liver, colon and Zymbal's gland carcinogenesis after initiation with diethylnitrosamine and 1,2-dimethylhydrazine

Combined effects of sodium nitrite (NaNO2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on liver, colon and Zymbal's gland carcinogenesis were assessed using a rat two-stage carcinogenesis model, with a focus on involvement of oxidative stress. Male 6-week-old F344 rats were given a single intraperitoneal injection of 200 mg/kg of diethylnitrosamine and 4 subcutaneous injections of 40 mg/kg of 1,2-dimethylhydrazine for initiation. Then, they were administered 0 or 300 ppm IQ in the diet or 0, 0.1 or 0.2% NaNO2 in their drinking water for 27 weeks. The treatment with NaNO2+IQ significantly enhanced colon and Zymbal's gland carcinogenesis and tended to enhance hepatocarcinogenesis. The incidence of lung tumors in the IQ-treated groups was significantly increased as compared with the initiation alone group. In a second experiment, male rats were given IQ or NaNO2 under the same conditions as before for 1 week, and at sacrifice, their liver and colon tissue or mucosa were collected for analysis of 8-hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARS), acrolein-modified protein and the bromodeoxyuridine-labeling index (BrdU-LI) (in the colon). In the colon, 8-OHdG, acrolein-modified protein levels and BrdU-LI were significantly increased by the combined treatment. These results indicate that the treatment with NaNO2 enhances IQ-induced colon and Zymbal's gland carcinogenesis in rats and that oxidative DNA damage and lipid peroxidation may partly be involved, especially in the colon. In addition, this experiment showed that IQ can act as a potent lung carcinogen in rats.

Carcinogenicity and Chronic Toxicity of 1,4-Dichloro-2-nitrobenzene in Rats and Mice by Two Years Feeding

Carcinogenicity and chronic toxicity of 1,4-dichloro-2-nitrobenzene (DCNB) were examined by feeding each group of 50 F344 rats and 50 BDF1 mice of both sexes a DCNB-containing diet at a concentration of 0 (control), 320, 800 or 2,000 ppm (w/w) for 2 yr. In rats, incidences of hepatocellular adenomas and carcinomas and their combined incidence were increased in the 2,000 ppm-fed males, together with increased incidence of basophilic cell foci in the 800 and 2,000 ppm-fed males. A dose-related increase in combined incidences of renal cell adenomas and carcinomas was noted. Incidence of Zymbal gland adenomas tended to increase in the 2,000 ppm-fed males. In mice, incidences of hepatocellular adenomas in the 800 and 2,000 ppm-fed females and hepatocellular carcinomas in the 2,000 ppm-fed males and in the 800 and 2,000 ppm-fed females were increased. Incidence of hepatoblastomas was increased in all DCNB-fed males and in the 2,000 ppm-fed females. Signs of chronic toxicity were characterized by centrilobular hypertrophy of hepatocytes with nuclear atypia in mice, increased relative liver weight in rats, a dose-related increase in incidences of chronic progressive nephropathy with advanced grades of severity in male rats, and decreased hemoglobin concentration and hematocrit accompanied by increased bone marrow hematopoiesis in female rats. Carcinogenic activity of DCNB was evaluated for the three different tumors, and sensitive signs of the chronic toxicity were dis-

Chemically induced immunotoxicity in a medium-term multiorgan bioassay for carcinogenesis with Wistar rats

A variety of chemicals can adversely affect the immune system and influence tumor development. The modifying potential of chemical carcinogens on the lymphoid organs and cytokine production of rats submitted to a medium-term initiation-promotion bioassay for carcinogenesis was investigated. Male Wistar rats were sequentially initiated with N-nitrosodiethylamine (DEN), N-methyl- N-nitrosourea (MNU), N-butyl- N-(4hydroxybutyl)nitrosamine (BBN), dihydroxy-di- n-propylnitrosamine (DHPN), and 1,2-dimethylhydrazine (DMH) during 4 weeks. Two initiated groups received phenobarbital (PB) or 2-acetylaminofluorene (2-AAF) for 25 weeks and two noninitiated groups received only PB or 2-AAF. A nontreated group was used as control. Lymphohematopoietic organs, liver, kidneys, lung, intestines, and Zymbal's gland were removed for histological analysis. Interleukin (IL)-2, IL-12, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-10, and transforming growth factor beta1 (TGF-β1) levels were determined by ELISA in spleen cell culture supernatants. At the fourth week, exposure to the initiating carcinogens resulted in cell depletion of the thymus, spleen and bone marrow, and impairment of IL-2, IL-12, and IFN-γ production. However, at the 30th week, no important alterations were observed both in lymphoid organs and cytokine production in the different groups. The results indicate that the initiating carcinogens used in the present protocol exert toxic effects on the lymphoid organs and affect the production of cytokines at the initiation step of carcinogenesis. This early and reversible depression of the immune surveillance may contribute to the survival of initiated cells facilitating the development of future neoplasia.