Prenatal test cohort of a modified rat comparative thyroid assay adding brain thyroid hormone measurements and histology but lowering group size appears able to detect disruption by sodium phenobarbital.

The Comparative Thyroid Assay (CTA, USEPA) is a screening test for thyroid hormone (TH) disruption in peripheral blood of dams and offspring. Recently, we began investigating feasible improvements to the CTA by adding examination of offspring brain TH concentrations and brain histopathology. In addition, we hypothesize that the number of animals required could be reduced by 50 % while still maintaining sensitivity to characterize treatment related changes in THs. Previously, we showed that the prenatal test cohort of the modified CTA could detect 1000 ppm sodium phenobarbital (NaPB)-induced suppression of brain T3 (by 9 %) and T4 (by 33 %) with no significant changes in serum T3 and T4 (less than 8 %). In the current study we expanded the dose response in a prenatal test cohort. Pregnant SD rats (N = 10/group) were exposed to 0, 1000 or 1500 ppm NaPB in the diet from gestational days (GD) 6 to GD20. Serum THs concentrations in GD20 dams together with serum/brain THs concentrations and brain histopathology in the GD20 fetuses were examined. NaPB dose-dependently suppressed serum T3 (up to -26 %) and T4 (up to -44 %) in dams, with suppression of T3 in serum (up to -26 %) and brain (up to -18 %) and T4 in serum (up to -26 %) and brain (up to -29 %) of fetuses but without clear dose dependency. There were no remarkable findings that deviated significantly from controls in GD20 fetal brain by qualitative histopathology. Overall, the present study suggests that the prenatal test cohort of this modified CTA is able to detect the expected fetal TH disruptions by prenatal exposure to NaPB, while also reducing the number of animals used by 50 %, consistent with the results of our previous study. These findings add to the suggestion that lowering group sizes and adding endpoints may be a useful alternative to the original CTA design.

Feasibility study for a downsized comparative thyroid assay with measurement of brain thyroid hormones and histopathology in rats: Case study with 6-propylthiouracil and sodium phenobarbital at high dose.

Concern has been raised that thyroid hormone disruptors (THDs) may potentially interfere with the developing brain, but effects of mild suppression of maternal THs by environmental contaminants on neonatal brain development are not fully understood. The comparative thyroid assay (CTA) is a screening test for offspring THDs, but it requires several animals and is criticized that reliance on serum THs alone as predictive markers of brain malfunction is inadequate. To verify feasibility of the downsized CTA but additional examination of brain THs levels and histopathology, we commenced internal-validation studies. This paper presents the data of the study where 6-propylthiouracil (6-PTU, 10 ppm) and sodium phenobarbital (NaPB, 1000 ppm) were dosed by feeding from gestational days (GD)6-20, and from GD6 to lactation day 21. The modified CTA detected 6-PTU-induced severe (>70%) suppression of serum THs in dams, with >50% suppressed serum/brain TH levels in offspring and brain heterotopia in postnatal day 21 pups. The modified CTA also detected NaPB-induced mild (<35%) suppression of serum THs in dams, with mild (<35%) reduction of serum/brain TH levels in fetuses but not in pups. These findings suggest that the modified CTA may have a potential as a screening test for offspring THDs.

Evaluation of the mode of action and human relevance of liver tumors in male mice treated with epyrifenacil.

Epyrifenacil (trademark name: Rapidicil®), a novel protoporphyrinogen oxidase (PPO)-inhibiting herbicide, induces hepatocellular adenomas and carcinomas in male CD-1 mice after 78 weeks treatment. The mode of action (MOA) of these mouse liver tumors and their relevance to humans was assessed based on the 2006 International Programme on Chemical Safety (IPCS) Human Relevance Framework. Epyrifenacil is not genotoxic and induced liver tumors via the postulated porphyria-mediated cytotoxicity MOA with the following key events: (#1) PPO inhibition; (#2) porphyrin accumulation; (#3) hepatocellular injury; with (#4) subsequent regenerative cell proliferation; and ultimately (#5) development of liver tumors. This article evaluates the weight of evidence for this MOA based on the modified Bradford Hill criteria. The MOA data were aligned with the dose and temporal concordance, biological plausibility, coherence, strength, consistency, and specificity for a porphyria-mediated cytotoxicity MOA while excluding other alternative MOAs. Although the postulated MOA could qualitatively potentially occur in humans, we demonstrate that it is unlikely to occur in humans because of quantitative toxicodynamic and toxicokinetic differences between mice and humans. Therefore, this MOA is considered not relevant to humans, utilizing the IPCS Human Relevance Framework; consequently, a nonlinear, threshold dose response would be appropriate for human risk assessment.

Elucidation of the species differences of epyrifenacil-induced hepatotoxicity between mice and humans by mass spectrometry imaging analysis in chimeric mice with humanized liver.

Epyrifenacil, one of the protoporphyrinogen oxidase (PPO)-inhibiting herbicides, is hepatotoxic in rodents. Previous in vitro assays detected species differences in both kinetics (active hepatic uptake) and dynamics (PPO inhibitory activity) of S-3100-CA, which is a causal metabolite of the hepatotoxicity, suggesting that humans are less sensitive to the epyrifenacil-induced hepatotoxicity than are rats and mice. To elucidate the species differences in the epyrifenacil-induced hepatotoxicity between mice and humans simultaneously, this study fed epyrifenacil to chimeric mice with humanized liver with low replacement index of human hepatocytes. The distribution of S-3100-CA in the liver and subsequent protoporphyrin IX (PPIX) accumulation, an index of PPO inhibition, were compared between human and host mouse hepatocytes using mass spectrometry imaging (MSI) analysis of chimeric liver. The results showed that S-3100-CA and PPIX were significantly colocalized in regions of the liver slice containing host mouse hepatocytes, and thus it was suggested that epyrifenacil had significantly less effect on human livers than mouse livers because of the species differences in both kinetics and dynamics of S-3100-CA. Moreover, the hepatic uptake assay using cryopreserved primary hepatocytes of rats, mice and humans with inhibitors revealed that S-3100-CA is a substrate of organic anion transporting polypeptides (OATPs). These data corroborate the contribution of OATPs to hepatocellular uptake of S-3100-CA, especially in mice, and subsequent PPIX accumulation by more potent S-3100-CA-induced PPO inhibition in mice. MSI analysis of chimeric mice with humanized liver is a useful technique for elucidating species differences in pharmacokinetics and subsequent changes in toxicological biomarkers.

Club Cells Are the Primary Target for Permethrin-Induced Mouse Lung Tumor Formation.

Permethrin has been shown to increase lung adenomas in female CD-1 mice, but not in male mice or Wistar rats. The proposed mode of action (MOA) for permethrin-induced female mouse lung tumor formation involves morphological changes in Club cells; increased Club cell proliferation; increased Club cell hyperplasia, and lung tumor formation. In this study, the treatment of female CD-1 mice with tumorigenic doses (2500 and 5000 ppm) of permethrin, but not with a nontumorigenic dose (20 ppm), for 14 and/or 28 days increased Club cell replicative DNA synthesis. Global gene expression analysis of female mouse lung samples demonstrated that permethrin treatment up-regulated 3 genes associated with cell proliferation, namely aldehyde dehydrogenase 3a1 (Aldh3a1), oxidative stress-induced growth inhibitor 1, and thioredoxin reductase 1. Treatment with 2500 and 5000 ppm, but not 20 ppm, permethrin for 7 days produced significant increases in mRNA levels of these 3 genes. Immunohistochemical analysis demonstrated that Club cell secretory protein, CYP2F2, and ALDH3A1 colocalized in Club cells; confirmed by flow cytometry analysis of lung cells employing KI67 as a cell proliferation marker. Overall, the present data extend the proposed MOA by demonstrating that Club cells are the primary initial target of permethrin administration in female mouse lungs. As humans are quantitatively much less sensitive to agents that increase Club cell proliferation and lung tumor formation in mice, it is most likely that permethrin could not produce lung tumors in humans. This conclusion is supported by available negative epidemiological data from several studies.

Chimeric Mouse With Humanized Liver Is an Appropriate Animal Model to Investigate Mode of Action for Porphyria-Mediated Hepatocytotoxicity.

Porphyrinogenic compounds are known to induce porphyria-mediated hepatocellular injury and subsequent regenerative proliferation in rodents, ultimately leading to hepatocellular tumor induction. However, an appropriate in vivo experimental model to evaluate an effect of porphyrinogenic compounds on human liver has not been fully established. Recently, the chimeric mouse with humanized liver (PXB mice) became widely used as a humanized model in which human hepatocytes are transplanted. In the present study, we examined the utility of PXB mice as an in vivo experimental model to evaluate the key events of the porphyria-mediated cytotoxicity mode of action (MOA) in humans. The treatment of PXB mice with 5-aminolevulinic acid, a representative porphyrinogenic compound, for 28 days caused protoporphyrin IX accumulation, followed by hepatocyte necrosis, increased mitosis, and an increase in replicative DNA synthesis in human hepatocytes, indicative of cellular injury and regenerative proliferation, similar to findings in patients with porphyria or experimental porphyria models and corresponding to the key events of the MOA for porphyria-mediated hepatocellular carcinogenesis. We conclude that the PXB mouse is a useful model to evaluate the key events of the porphyria-mediated cytotoxicity MOA in humans and suggest the utility of PXB mice for clarifying the human relevancy of findings in mice.

Cell proliferation analysis is a reliable predictor of lack of carcinogenicity: Case study using the pyrethroid imiprothrin on lung tumorigenesis in mice.

In the mouse carcinogenicity study, an apparent increase in lung adenocarcinoma was observed in male mice at 7000 ppm. Based on the overall evaluation of toxicology, oncology, pathology and statistics, we concluded that the apparent increase in lung tumors is not relevant for evaluation of carcinogenicity of imiprothrin (Regul Toxicol Pharmacol, 105, 1-14, 2019). To investigate whether imiprothrin has any mitogenic effect on mouse Club cells, the present study examined its effects on replicative DNA synthesis of Club cells and lung histopathology in male mice treated with imiprothrin for 7 days at 3500 and 7000 ppm in the diet. Isoniazid, a known mouse lung mitogen and tumor inducer, was also examined at 1000 ppm in the diet as a positive control of Club cell mitogenesis and morphological changes. Neither imiprothrin nor isoniazid caused any necrotic changes in lung by light or electron microscopy. There were no increases observed in the bromodeoxyuridine (BrdU) labeling index in the imiprothrin groups, while there was a statistically significant increase in the BrdU labeling index in the isoniazid group. These findings demonstrate that imiprothrin does not induce mouse Club cell proliferation or morphologic changes, supporting our previous conclusion described above. Thus, imiprothrin should not be classified as a carcinogen. Furthermore, this study indicates that short-term studies focusing on cell proliferation can be reliable for predicting a lack of carcinogenic potential of test chemicals.

Segmental aplasia of a uterine horn in two RccHan:WIST rats.

Aplasia of the uterine horn caused by developmental defects has been reported in several species but has not been reported in RccHan:WIST rats. We encountered spontaneous segmental aplasia of a right uterine horn in two RccHan:WIST rats and detailed its pathological characteristics. The right uterine horn of both rats had similar gross and histological appearances. At necropsy, there was segmental loss of the tissues corresponding to normal right uterine horn, which consisted of a fibrous band connected to the uterine cervix. A cystic structure with clear and colorless fluid was observed in the cranial segment of the right uterine horn close to the right oviduct. The cystic structure was thought to be a partially developed tissue to the right uterine horn. The cystic structure seemed to be derived from the right uterine horn. Histologically, a single layer of cuboidal epithelium lined the luminal surface of the cystic structure, the endometrium was thin, and no uterine glands were observed. The fibrous band was composed of α-SMA positive smooth muscle cells, connective tissue, and blood vessels, but cytokeratin AE1/AE3 positive epithelium and uterine endometrium were absent. Based on these gross findings and histological features, segmental aplasia of a uterine horn was diagnosed. To our knowledge, these cases of segmental aplasia of a uterine horn are the first ones described in RccHan:WIST rats.

The effects on the endocrine system under hepatotoxicity induction by phenobarbital and di(2-ethylhexyl)phthalate in intact juvenile male rats.

Phenobarbital (PB) and Di (2-ethylhexyl) phthalate (DEHP), an anti-epileptic drug and a plasticizer used in flexible polyvinylchloride formulations, respectively, are well-known typical hepatotoxicants. This study investigated the effects of PB (100 mg/kg/day) or DEHP (500 mg/kg/day) on the endocrine system in intact juvenile/peripubertal male rats exposed for 31 days beginning on postnatal day 23. Slight hormone level changes, histopathological changes in thyroid gland or induction of UDP-glucuronosyltransferase in liver were observed in both the PB and DEHP groups. One of the assumed mechanisms inducing thyroid effects is predictable to be secondary changes based on the enhancement in thyroid hormone metabolism via the induction of hepatic microsomal enzymes. No reproductive system-related changes in organ weights, histopathology, and sexual maturation were observed in both groups. Lower testosterone level was observed in the PB group. CYP2B and CYP3A, which are involved in testosterone metabolism, were induced in liver of the PB group. There was no change of 17ß-hydroxysteroid dehydrogenase activity in testis of both groups. Lower testosterone level in the PB-treated male rats was attributed to an indirect, hepatotoxicity-associated effect on the reproductive system and not to direct effects on testis such as the antiandrogenic activity and the inhibition of steroidogenesis. These results did not indicate that PB or DEHP exposure affects the endocrine system directly.

An Evaluation of the Human Relevance of the Lung Tumors Observed in Female Mice Treated With Permethrin Based on Mode of Action.

Permethrin increased the incidence of bronchiolo-alveolar adenomas in female mice but not male mice or female or male rats. Studies were conducted to determine whether permethrin has mitogenic activity in Club cells in mouse lung as the basis for the mode of action (MOA) for the lung adenoma induction. Several short-term experiments focusing on time-course, dose-response, reversibility, sex difference, strain difference, and species difference were evaluated for Club cell proliferation and morphology. The findings demonstrated that permethrin slightly and continuously enhanced Club cell proliferation at tumor-associated dose levels in female mice, but did not increase proliferation in male mice or in female rats. Electron microscopic examination demonstrated that permethrin produced morphological alterations in Club cells prior to increasing the Club cell proliferation. There was no evidence of increased cell death. These alterations in Club cells were also observed with a close structural analog cypermethrin. Taken together, the present studies provide evidence that the MOA for induction of mouse lung adenomas by permethrin involves slight morphological effects on Club cells, sustained Club cell proliferation, and eventually hyperplasia and bronchiolo-alveolar adenoma in susceptible mice. The potential human carcinogenic hazard of permethrin based on the tumorigenic MOA for lung tumors in mice was evaluated using the International Programme on Chemical Safety Human Relevance Framework. As humans are quantitatively much less sensitive to agents that increase Club cell proliferation and tumor formation in mice, it is not likely permethrin will lead to an increase in susceptibility to lung tumor development in humans. Epidemiological data for permethrin strongly supports this conclusion.

Alteration of microRNA expressions in the pons and medulla in rats after 3,3'-iminodipropionitrile administration.

Although 3,3'-iminodipropionitrile (IDPN) is widely used as a neurotoxicant to cause axonopathy due to accumulation of neurofilaments in several rodent models, its mechanism of neurotoxicity has not been fully understood. In particular, no information regarding microRNA (miRNA) alteration associated with IDPN is available. This study was conducted to reveal miRNA alteration related to IDPN-induced neurotoxicity. Rats were administered IDPN (20, 50, or 125 mg/kg/day) orally for 3, 7, and 14 days. Histopathological features were investigated using immunohistochemistry for neurofilaments and glial cells, and miRNA alterations were analyzed by microarray and reverse transcription polymerase chain reaction. Nervous symptoms such as ataxic gait and head bobbing were observed from Day 9 at 125 mg/kg. Axonal swelling due to accumulation of neurofilaments was observed especially in the pons, medulla, and spinal cord on Day 7 at 125 mg/kg and on Day 14 at 50 and 125 mg/kg. Furthermore, significant upregulation of miR-547* was observed in the pons and medulla in treated animals only on Day 14 at 125 mg/kg. This is the first report indicating that miR-547* is associated with IDPN-induced neurotoxicity, especially in an advanced stage of axonopathy.

Optimal dose selection of N-methyl-N-nitrosourea for the rat comet assay to evaluate DNA damage in organs with different susceptibility to cytotoxicity.

The in vivo rodent alkaline comet assay (comet assay) is a promising technique to evaluate DNA damage in vivo. However, there is no agreement on a method to evaluate DNA damage in organs where cytotoxicity is observed. As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the comet assay, we examined DNA damage in the liver, stomach, and bone marrow of rats given three oral doses of N-methyl-N-nitrosourea (MNU) up to the maximum tolerated dose based on systemic toxicity. MNU significantly increased the % tail DNA in all the organs. Histopathological analysis showed no cytotoxic effect on the liver, indicating clearly that MNU has a genotoxic potential in the liver. In the stomach, however, the cytotoxic effects were very severe at systemically non-toxic doses. Low-dose MNU significantly increased the % tail DNA even at a non-cytotoxic dose, indicating that MNU has a genotoxic potential also in the stomach. Part of the DNA damage at cytotoxic doses was considered to be a secondary effect of severe cell damage. In the bone marrow, both the % tail DNA and incidence of micronucleated polychromatic erythrocytes significantly increased at non-hematotoxic doses, which were different from the non-cytotoxic doses for liver and stomach. These findings indicate that an optimal dose for detecting DNA damage may vary among organs and that careful attention is required to select an optimum dose for the comet assay based on systemic toxicity such as mortality and clinical observations. The present study shows that when serious cytotoxicity is suggested by increased % hedgehogs in the comet assay, histopathological examination should be included for the evaluation of a positive response.

Genotoxicity evaluation of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate using a combined rat comet/micronucleus assays.

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo alkaline comet assay (comet assay), we examined DNA damage in the liver, stomach, and bone marrow of rats dosed orally three times with up to 2000 mg/kg of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate. All three compounds gave negative results in the liver and stomach. In addition, a bone marrow comet and micronucleus analysis revealed that benzene, but not di(2-ethylhexyl) phthalate or trisodium ethylenediamine tetraacetic acid monohydrate induced a significant increase in the median % tail DNA and micronucleated polychromatic erythrocytes, compared with the respective concurrent vehicle control. These results were in good agreement with the previously reported genotoxicity findings for each compound. The present study has shown that combining the micronucleus test with the comet assay and carrying out these analyses simultaneously is effective in clarifying the mechanism of action of genotoxic compounds such as benzene.

Circulating miR-9* and miR-384-5p as potential indicators for trimethyltin-induced neurotoxicity.

Circulating microRNAs (miRNAs) show promise as biomarkers due to their tissue-specific expression and high stability. This study was conducted to investigate whether nervous system-enriched miR-9* and hippocampus-enriched miR-384-5p could be indicators of neurotoxicity in serum. Rats were given a single administration of trimethyltin (TMT) chloride at 6, 9, or 12 mg/kg by gavage, and brain and serum were collected 1, 4, and 7 days after administration. MiR-9* and miR-384-5p levels in serum and hippocampus were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), and their neurotoxicity detection sensitivities were compared with nervous symptoms, auditory response, and histopathology. TMT caused tremor, hypersensitivity, and decreased auditory response at 12 mg/kg on day 1 and at 9 mg/kg on day 4. Histopathologically, neural cell death and glial reaction were observed in brain (mainly hippocampus) at 12 mg/kg on day 1, 4, and 7 and at 6 and 9 mg/kg on day 4 and 7. MiR-9* and miR-384-5p levels were elevated in serum at 9 and 12 mg/kg on days 4 and 7 (at 9 mg/kg on day 7, miR-9* only) but were not changed in hippocampus. These miRNAs were considered to be elevated with the evolution of neural cell death and were thus considered possible novel indicators of neurotoxicity.

A Spontaneous Oligodendroglioma in the Lumbar Portion of the Spinal Cord in a Young BrlHan:WIST@Jcl (GALAS) Rat.

Oligodendroglioma is a rare tumor originating from oligodendrocytes found mainly in the cerebrum in aged rats. Only a few reports have shown spontaneous occurrence of this tumor in the spinal cord, and no report has mentioned its occurrence in young rats. We encountered a case of spontaneous oligodendroglioma in the lumbar portion of the spinal cord in a young (9 weeks old) female BrlHan:WIST@Jcl (GALAS) rat. Here we report the detailed histopathological and immunohistochemical characteristics of this case. No clinical signs, no gross lesions at necropsy, and no specific changes in hematology or blood biochemistry were observed. The tumor was located in the dorsal funiculus in the lumbar portion of the spinal cord and widely spread to the dorsal root nerve. The neoplastic cells showed a sheet-like growth pattern and had small round nuclei, clear cytoplasm and distinct cell borders that resulted in a honeycomb pattern. No mitotic figures or other histological lesions were observed. The neoplastic cells were positively stained for Olig2 and PCNA. The present case was considered to be a low-grade oligodendroglioma based on the histological and immunohistochemical features. To our knowledge, our case is considered to be extremely rare and the first report in a young rat.

Characterization of rat testicular teratoma and its derived cell lines, with particular reference to possible mesenchymal differentiations.

The original tumor, 4 cm in diameter, was found in the left testis of a 2-month old SD rat. The tumor consisted of well-differentiated, mature tissues such as bone, cartilage, adipose tissue, smooth and skeletal muscles, skin, hair, glands (salivary, sebaceous, apocrine and pancreatic exocrine glands) and trachea, as well as nerve tissues. The tumor was diagnosed as a mature type of teratoma, a rare in rat testis. Cloned cell lines (named TSD-B4S and TSD-F9R) were established from the tumor; cellular properties of these cell lines were similar to each other; basically, their cultured cells exhibited vimentin-positive mesenchymal nature with occasional cells reacting to α-smooth muscle actin, glial fibrillary acidic protein and CD163 (a macrophage marker). The cell lines showed tumorigenicity when inoculated into nude mice, being composed of immature mesenchymal cells arranged mainly in a sheet. In TSD-B4S cells treated with differentiation factors, we demonstrated mesenchymal differentiations towards adipogenic, osteogenic and myofibrogenic cells. The cell line (TSD-B4S) would become a useful tool for studies on stem cell differentiation, because the teratoma arises from primordial germ cells like embryonic stem cells.

Well-differentiated teratoma in a mouse uterus.

Teratomas commonly occur in the testis and ovary, whereas in the uterus they are rare. The authors report findings for a mass detected in the uterus of a 26-week-old mouse in a colony of C57BL/6 bred in their laboratory. The mass was located in the endometrium and protruded into the lumen. Histopathologically, it consisted of abnormal diploblastic or triploblastic tissues. Bone with a growth plate and myeloid cells, as well as cartilage, was mainly observed. It also included melanocytes, exocrine gland-like cells, striated muscle, and neuron-like cells. While these tissues were accompanied by extensive necrosis, all of them were well differentiated and lacked features of malignancy, such as invasion and metastasis. This mouse had experienced parturition, but fetal tissue was not observed in the lesion. Therefore, the lesion was diagnosed as a benign teratoma, which was spontaneously developed in the uterus.

Circulating microRNAs, possible indicators of progress of rat hepatocarcinogenesis from early stages.

MicroRNAs (miRNAs), a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level, are believed promising biomarkers for several diseases as well as a novel target of drugs, including cancer. In particular, miRNAs might allow detection of early stages of carcinogenesis. The present study was conducted to provide concrete evidence using chemical-induced hepatocarcinogenesis in rat as a model. We thereby observed aberrant fluctuation of circulating miRNAs in the serum of rats not only with neoplastic lesions such as hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC), but also with preneoplastic lesions, such as foci of hepatocellular alteration (FHA). Additional qRT-PCR analysis revealed gradual elevation of some circulating miRNAs (i.e., let-7a, let-7f, miR-34a, miR-98, miR-331, miR-338 and miR-652) with progress of hepatocarcinogenesis. Interestingly, increased levels of let-7a, let-7f and miR-98 were statistically significant even in the serum of rats at very early stages. These findings provide the first evidences that circulating miRNAs have the potential to predict carcinogenesis at earlier stages, preneoplastic lesions than with previous biomarkers and that they might be utilized to monitor the progress of tumor development.

Ethanol Does Not Promote MeIQx-initiated Rat Colon Carcinogenesis Based on Evidence from Analysis of a Colon Cancer Surrogate Marker.

Epidemiological studies suggest that alcohol consumption increases the risk of developing colorectal cancer. However, the data are confounded by numerous cosegregating variables. To cast further light on the relationships between alcohol intake and colon cancer development, 21-day-old male F344/DuCrj rats were fed 200 ppm 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and doses of 0, 0.1, 0.3, 1, 3, 10 and 20% of ethanol in their drinking water ad libitum for 16 weeks thereafter. The rats were sacrificed after 24 weeks of experiment, and aberrant crypt foci (ACF), surrogate lesions for colon cancer, were examined under a light microscope at low magnification. Ethanol was found not to affect the ACF formation at any dose compared with the initiated-controls. Furthermore, ethanol did not alter colon epithelial cell proliferation. These data, obtained by analysis of a colon cancer surrogate marker lesion, indicate that ethanol lacks promotion activity for MeIQx-initiated rat colon carcinogenesis.