In Utero Exposure to 3,3',4,4', 5-Pentachlorobiphenyl Dose-Dependently Induces N-butyl-4-(hydroxybutyl) Nitrosamine in Rats With Urinary Bladder Carcinoma.

Polychlorinated biphenyls (PCBs) are fat-soluble environmental pollutants that can accumulate in adipose tissue or be secreted in milk. N-butyl-4-(hydroxy butyl) (BBN), a rat bladder carcinogen, recruits the host metabolism to yield its ultimate carcinogenic form via CYP1s. Since estrogen receptors (ERs) mediate biological responses important for the growth of bladder carcinoma, we investigated PCNA, Cyclin D1, ERs, CYP1s, and AhR expression in BBN rat bladder carcinomas with prenatal PCB exposure. Female SD rats were treated with 7.5 µg, 250 ng, and 2.5 ng of 3,3',4,4',5-pentachlorobiphenyl (PCB126)/kg or vehicle on days 13 to 19 post-pregnancy. Six-week-old male offspring were treated with 0.05% BBN for 10 weeks before being anesthetized and the urinary bladder wall incised to expose the bladder carcinomas. N-butyl-4-(hydroxybutyl) bladder carcinoma incidence increased with prenatal PCB exposure dose-dependently. In bladder carcinoma, PCB126 exposure significantly increased PCNA, D1, ERα, CYPIA1, CYP1B1, and AhR expression dose-dependently, and increased ERα expression was particularly prominent. However, the expression of ERß was low, independent of the volume of PCB126 given, indicating similarity to the Vehicle group. We conclude that prenatal PCB126 exposure in rats can induce PCB126 to dose-dependently metabolize BBN via CYP1A1, and contribute to bladder carcinogenesis with upregulation of ERα expression.

Prenatal exposure to di(n-butyl) phthalate delays the spermatogenic cycle in rats: Investigation using a BrdU-injection method.

Di(n-butyl) phthalate (DBP) esters are plasticizers that are used to provide transparency and flexibility in household plastic products but can easily leach out to contaminate organisms and the environment. We investigated whether prenatal DBP exposure affects spermatogenesis in rats. Pregnant Sprague-Dawley rats were injected with DBP 10, 50, and 100 mg/kg, or vehicle, administered intragastrically, on gestation days 12-21. At 9 or 17 weeks, 5-bromodeoxyuridine (BrdU) 50 mg/kg was injected intraperitoneally, and one testis was removed 3 h later. The remaining testis was excised 12.95 days + 3 h after the BrdU injection. Immunohistochemical analysis of BrdU was performed with periodic acid-Schiff and hematoxylin counterstaining for a quantitative analysis of the delay in one cycle of spermatogenesis. The DBP 100 mg group showed that the ratio of the appearance of seminiferous tubules in stages VII and VIII were significantly decreased, but those of stages IX and X were significantly increased compared to the Vehicle group. The reference value for the duration of spermatogenesis per cycle was set at 310.8 h. The DBP 100 mg group showed a significant delay in the duration of one cycle of spermatogenesis (16.95 h at puberty and 19.01 h at adulthood) compared with the Vehicle group. This study determined that F1-generation rats with prenatal DBP 100 mg exposure revealed significant accumulation of spermatogenic cells at stages IX to X in the second and third cycles, and the significant delay in the duration of spermatogenesis was more prominent at adulthood than in puberty.

Sertoli cells proliferate in adult rats with prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl.

Sertoli cells play a critical role in spermatogenesis, and in adults, they are terminally differentiated with loss of proliferative activity. This study revealed Sertoli cell proliferation in 17-week-old Sprague-Dawley rats whose dams had been intragastrically administered 250 ng of 3,3',4,4',5-pentachlorobiphenyl/kg on days 13-19 postconception. Immunohistochemical evidence of proliferating cell nuclear antigen (PCNA) expression and electron microscope observation of mitotic figures confirmed the proliferation. Because the serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone concentrations were similar to those of vehicle-treated rats, a direct endocrine cause for the observed effects was unlikely.

Cyclin D1/cdk4, estrogen receptors α and ß, in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric carcinogenesis: immunohistochemical study.

Hyperproliferative cell growth due to cyclin D1/cdk4, marker of cellular proliferation, is considered to be regulated by the expression of estrogen receptors (ERs). We investigated the immunohistochemical expression of cyclin D1/cdk4 and ERs in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced rat gastric carcinogenesis. The gastric cancer incidence and expression of cyclin D1/ckd4 in gastric carcinogenesis were significantly higher in males than females. Although the ERα expression index was similar in both sexes, the ERß expression in preneoplastic hyperplastic lesions as well as gastric cancers was significantly higher in females than in males. The present study revealed a gender difference in MNNG-induced rat gastric carcinogenesis that seemed to involve the sex difference in cyclin D1/cdk4 expression, and ERß expression became evident at the preneoplastic promotion stage in gastric carcinogenesis.

Sex-associated difference in estrogen receptor ß expression in N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric cancers in rats.

Epidemiologic studies indicate that the incidence of gastric cancer is higher in males than in females. Although the mechanisms mediating this difference are unclear, a role for estrogens has been proposed. We used Western blotting to evaluate the role of estrogen receptor (ER) subtypes ERα and ERß and proliferating cell nuclear antigen (PCNA) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats; ERα and ERß mRNA levels also were analyzed by quantitative real-time RT-PCR analysis. The incidence of gastric cancer was significantly higher in male than female rats. In both sexes, ERα expression was similar in MNNG-treated cancerous and noncancerous tissues and normal gastric tissue. However, ERß expression in MNNG-treated cancerous and noncancerous tissues was significantly lower in male rats and higher in female rats than that in normal gastric tissue; MNNG-induced cancerous tissue showed the highest ERß expression. PCNA expression in MNNG-treated cancerous tissues was higher than that in noncancerous tissues, and was higher in male rats than female rats. Western blotting results were consistent with the mRNA changes determined by quantitative real-time RT-PCR. The present study provides evidence of a sex-associated difference in ERß and PCNA expression in MNNG-induced gastric cancers in Wistar rats.

Testicular spermiation failure in rats exposed prenatally to 3,3',4,4',5-pentachlorobiphenyl.

Testicular spermatogenesis was studied in 7-, 10-, 13- and 17-week-old Sprague-Dawley rats whose dams had been administered intragastrically with 2.5, 25, or 250 ng of 3,3',4,4',5-pentachlorobiphenyl (PCB126) or vehicle on days 13-19 of gestation. The 250 ng groups among the 7-, 10- and 13-week-old offspring showed significant inhibition of mature spermatid release (spermiation), but 17-week-old offspring did not show this. These alterations were not observed in other PCB126 and vehicle groups, and no germ cell or Sertoli cell degeneration were observed in any group. Spermiation failure at puberty appeared in those rats born to dams exposed 250 ng/kg PCB126 on days 13-19 of gestation was reversible change that recovered at adulthood. Because the serum testosterone, luteinizing hormone and follicle-stimulating hormone concentrations were similar in the PCB126 and vehicle groups, a direct endocrine cause for the observed effects was unlikely.

L-type amino-acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer.

To find reliable biomarkers for high-grade malignancy, the relationship between immunohistochemical L-type amino-acid transporter 1 (LAT1) expression of biopsy samples, determined with the newly developed monoclonal antibody against human LAT1, and prognosis of patients with prostate cancer, was investigated. The intensity and score of immunohistochemical LAT1 expression of first biopsy samples were assessed using the modified Sinicrope et al. method and were found to be correlated with poor survival for the study group of 114 surgically treated patients as a whole (P = 0.0002 and 0.0270, respectively). LAT1 intensity further had a significant relationship (P = 0.0057) with prognosis in pathological T3 + T4 groups. Multivariate analysis indicated that the LAT1 intensity and score were more reliable prognostic markers, compared with the Gleason score and the Ki-67 labeling index. A relationship of the LAT1 intensity and score with prognosis could also be confirmed in 63 patients with inoperable cancer (P = 0.0070 and <0.0001, respectively). Similarly, significant differences in prognosis were confirmed in clinical T3 + T4 groups (P = 0.0091 and 0.0244, respectively). Moreover, the combination of LAT1 expression and Gleason score was found to have a more reliable correlation with prognosis. Thus, elevated LAT1 expression in prostate cancers is a novel independent biomarker of high-grade malignancy, which can be utilized together with the Gleason score, which is mainly dependent on cellular and structural atypia, to assess prognosis.

Sertoli-Leydig cell tumor of the testis in a Sprague-Dawley rat.

A rare intratubular gonadal stromal tumor was present in the testis of a 7-wk-old male Sprague-Dawley rat. The tumor comprised an intratubular mixture of 2 types of tumor cells with intercellular junctions: the predominant tumor cells were consistent with a Sertoli cell origin, and cells comprising the minor population were situated on basolateral side of the tubuli, consistent with a Leydig cell origin. The neoplastic Sertoli cells had large pleomorphic nuclei and clear cytoplasm with many tubulovesicular cristae and free ribosomes, whereas the neoplastic Leydig cells showed relatively small pleomorphic nuclei, dark cytoplasm with rich smooth endoplasmic reticulum, numerous mitochondria, and lipid droplets. Occasionally, a few transitional type neoplastic cells were observed. The presence of a thick or multilayered basement membrane was confirmed except in tumor-infiltrative lesions. The present case was considered to be a testicular mixed tubular Sertoli-Leydig cell tumor in a Sprague-Dawley rat.

Spermatogenesis in aged rats after prenatal 3,3',4,4',5-pentachlorobiphenyl exposure.

We previously reported that prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) had dose-related adverse effects on the spermatogenesis of 7(pubescent)- and 17(adult)-week-old rats, but the effects in middle and old age have been unclear. In this study, the spermatogenesis of male Sprague-Dawley rats whose dams had been injected (i.g.) with 25 pg, 2.5 ng, 250 ng, or 7.5 microg of PCB126/kg or the vehicle on days 13-19 post-conception was investigated at 52 and 90 weeks of age. At 52 weeks, the 7.5 microg group showed a significant decrease of preleptotene spermatocytes in stages VII-VIII seminiferous tubules, round spermatids increased at stages VI-VII and elongated spermatids decreased at stage VIII, while the spermatogenesis of the other PCB-treated groups were similar to that of the vehicle group. At 90 weeks, the 7.5 microg group showed a significant decrease of spermatogenic cells at many stages, and the 250 ng group showed a significant decrease of preleptotene spermatocytes at stages VII-VIII, and round spermatids increased at stages VI-VII, elongated spermatids decreased at stage VIII, and the spermatogenesis of the 2.5 ng and 25 pg groups were similar to those of the vehicle group. The present study showed that prenatal PCB126 exposure had dose-related adverse effects on spermatogenesis in aging rats and may have accelerated spermatogenic senescence. Because the serum testosterone levels of the PCB126 groups and the vehicle group were similar, a direct endocrine cause for the observed effects was unlikely.

Localization of Ang-1, -2, Tie-2, and VEGF expression at endothelial-pericyte interdigitation in rat angiogenesis.

Endothelial cells and pericytes play critical role in angiogenesis, which is controlled, in part, by the angiopoietin (Ang)/Tie-2 system and vascular endothelial growth factor (VEGF). Here, we investigated Ang, Tie-2, and VEGF expression within endothelial cells and pericyte interdigitations (EPI), which consist of cytoplasmic projections of pericytes and corresponding endothelial indentations. After subcutaneous implantation of a thermoreversible gelation polymer disc in rats, the capillary density was low on day 5, increased to a peak on day 7, and then decreased on days 10-20. A small number of EPI were observed on day 5, then increased sharply to a peak on day 10, but had decreased on day 20. Light and electron microscopy immunohistochemical and RNA in situ hybridization analyses revealed that Tie-2 localized at endothelial cells, and Ang-2 localized at endothelial cells and pericytes, while Ang-1 and VEGF localized at pericytes, and Ang-1 was most intensely observed at EPI of pericytes. Conventional quantitative RT-PCR and Western blot analyses revealed that the level of Ang-1 was low on days 5-7, then increased on days 10-20, while the level of VEGF was high on days 5-10, but had decreased on day 20. The level of Ang-2 remained high and Tie-2 remained at the level of the control on days 5-20. The present study showed that the angiogenic phase might be initiated by increases in Ang-2 and VEGF, while the microvessel maturation phase might be initiated by a relative increase in Ang-1 and a decrease in VEGF. Moreover, EPI might serve as a pathway for the Ang-1/Tie-2 system, with VEGF promoting pericyte recruitment for microvascular integrity.

Prenatal 3,3',4,4',5-pentachlorobiphenyl exposure modulates induction of rat hepatic CYP 1A1, 1B1, and AhR by 7,12-dimethylbenz[a]anthracene.

We previously reported the finding that prenatal exposure to a relatively low dose of PCB126 increases the rate of DMBA-induced rat mammary carcinoma, while a high dose decreased it. One of the most important factors determining the sensitivity to mammary carcinogenesis is the metabolic stage at administration of the carcinogenic agent. DMBA is a procarcinogen that recruits the host metabolism to yield its ultimate carcinogenic form, and CYP1A1 and CYP1B1 (CYP1) conduct this metabolism. We investigated the hepatic expression of CYP1 and AhR following oral administration of DMBA (100 mg/kg b.w.) (i.g.) to 50-day-old female Sprague-Dawley rats whose dams had been treated (i.g.) with 2.5 ng, 250 ng, 7.5 microg of PCB126/kg or the vehicle on days 13 to 19 post-conception. Real-time quantitative RT-PCR analysis revealed that the prenatal exposure to a relatively low dose of PCB126 (the 250 ng group) prolonged the higher expression of CYP1A1, CYP1B1, and AhR mRNA, while prenatal exposure to a high dose of PCB126 (the 7.5 microg group) prolonged the higher expression of CYP1A1 and AhR mRNA. Western blotting and immunohistochemical analyses were consistent with mRNAs changes. Because DMBA oxidation produces a highly mutagenic metabolite and is finally catalyzed by CYP1B1, a relatively low PCB126 dose might produce the biological character to potentially increase the risk of DMBA-induced mammary carcinoma.

CYP1 and AhR expression in 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma of rats prenatally exposed to 3,3',4,4',5-pentachlorobiphenyl.

We previously reported the finding that prenatal exposure to a relatively low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) acted as an enhancing agent for 17-beta-estradiol (E2)-dependent 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreased it. E2 is a known risk factor for mammary carcinoma, and CYP1A1 and 1B1 (CYP1) are the major enzymes catalyzing 2- and 4-hydroxylation of E2, respectively. We investigated the induction of CYP1 and aryl hydrocarbon receptor (AhR) in DMBA-induced mammary carcinoma using female Sprague-Dawley rats whose dams had been treated (i.g.) with 2.5 ng, 250 ng, 7.5 microg of PCB126/kg or the vehicle on days 13-19 post-conception. Immunohistochemical analysis revealed that the mammary carcinoma of the 250 ng group showed a significantly higher number of nuclei expressing estrogen receptor alpha (ER) and proliferating cell nuclear antigen (PCNA) compared to those of the other groups. Quantitative real-time RT-PCR analysis revealed that the 7.5 microg group showed a significantly higher level of CYP1A1 mRNA, and that the 250 ng group showed significantly higher levels of CYP1B1 mRNA. The level of AhR mRNA was significantly higher in both the 7.5 microg and 250 ng groups. Western blotting analysis was consistent with mRNA changes. It has been revealed that CYP1B1 catalyzes a step in the formation of 4-hydroxylated E2 metabolites, which show quite high mammary carcinogenicity. This study indicates that the enhancement of DMBA-induced mammary carcinogenicity in a relatively low PCB126 dose group might partially involve the higher expression of CYP1B1 and AhR in these carcinomas.

Molecular pathological analysis on the mechanism of liver carcinogenesis in dicyclanil-treated mice.

To investigate the mechanism of hepatocarcinogenesis due to dicyclanil (DC), an insect growth regulator for sheep, histopathological and molecular biological analyses were performed in the liver of male ICR mice fed on a diet containing 1500 ppm of DC for 2 weeks (Experiment I; Exp. I). In gene expression analyses using a large-scale cDNA microarray and RT-PCR, fluctuations of expressions of metabolism-/oxidation-/reduction-related genes, such as CYP1A, aldehyde dehydrogenase family 1 subfamily A1 (Aldh1a1), and thioredoxin reductase 1 (Txnrd1), were predominantly observed in the liver of the DC-treated group. In Experiment II (Exp. II), small-scale and metabolism/oxidative stress-specific cDNA microarray, real-time RT-PCR, and measurement of NF-kappaB protein were performed in the mice liver using a two-stage hepatocarcinogenesis model, in which the male ICR mice were fed on a diet containing 1500 ppm of DC for 7 weeks after a single injection of dimethylnitrosamine (DMN). These mice were subjected to two-thirds partial hepatectomy (PH) at week 3. During histopathological examinations, a remarkable increase in gamma-glutamyltransferase-positive cells was observed in the DMN+DC+PH group. During the microarray and PCR analyses, the metabolism and oxidative stress-related genes, such as Cyp1a, P450 oxidoreductase (Por), and thioredoxin reductase 1 (Txnrd1); a few DNA damage/repair genes, such as 8-oxoguanine DNA-glycosylase 1 (Ogg1); and growth arrest and DNA-damage-inducible 45 alpha (Gadd45a), were fluctuated in this group, together with a slight increase in the concentration of activated NF-kappaB. These results suggest that DNA damages due to oxidative stress may be involved in the mechanism of DC-induced hepatocarcinogenesis in mice.

Analysis of gene expression profiles of forestomach tumors in rasH2 mice initiated with N-ethyl-N-nitrosourea.

To clarify the mechanisms underlying enhancement of carcinogenesis in transgenic mice carrying a human prototype c-Ha- ras gene (rasH2 mouse), animals received a single intraperitoneal injection of 120 mg/kg N-ethyl-N-nitrosourea (ENU) and at 20 weeks thereafter expression profiles in three induced forestomach squamous cell carcinomas were assessed using high-density oligonucleotide microarrays. In addition, the reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess mRNA expression of human c-Ha- ras gene and some molecules involved in the Ras-regulated mitogen-activated protein kinase (MAPK) pathway. Compared with normal forestomach tissue from control mice, 416 and 368 genes, respectively, were found to be commonly up- and down-regulated by 2-fold or more in the three tumors. Many genes involved in tumor invasion and metastasis such as transforming growth factor beta1 and matrix metalloproteinases were up-regulated, reflecting tumor progression. RT-PCR analysis confirmed up-regulation of transgene, mouse endogenous Ha- ras, N- ras, raf, Mekk2, c- fos, junB, c- myc and cyclin D1. These results suggest that activation of the Ras-MAPK cascade following up-regulation of both human and mouse endogenous ras genes is involved in the enhanced tumorigenesis of ENU-induced forestomach squamous cell carcinomas in rasH2 mice.

Ser217Leu polymorphism of the HPC2/ELAC2 gene associated with prostatic cancer risk in Japanese men.

The HPC2/ELAC2 gene may be associated with hereditary/familial prostate cancer (PCa). Two common missense variants (Ser217Leu and Ala541Thr) have been reported in the gene. We performed mutational, allelotyping and expression analyses and a molecular epidemiological study to clarify the relations between this gene and prostatic diseases, including PCa and benign prostatic hyperplasia (BPH) in Japanese men. We screened for mutations in 109 patients with PCa including 11 patients from 1 hereditary and 9 familial PCa. Loss of heterozygosity and expression were analyzed. An epidemiological study was done in sporadic PCa (n=98) and BPH (n=143) using 1 novel (Ser627Leu) and 2 previously described polymorphisms of the HPC2/ELAC2 gene. Somatic or germline mutations were not confirmed in any cases of PCa. Loss of heterozygosity at 2 microsatellites, D17S1289 and D17S520, was detected in 1 of 38 and 1 of 35 cases, respectively. Expression analysis revealed decreased or absent mRNA expression in 6 of 38 tumors. Epidemiologic analysis showed that a Leu allele at codon 217 was significantly more frequent in patients with PCa than in controls (10.2% vs. 3.5%, odds ratio = 3.11; 95% confidence interval, 1.22-7.90). At codon 541, all patients with PCa or BPH and all control subjects had the Ala/Ala genotype. At codon 627, the incidence of the Leu variant was slightly, but not significantly, higher in patients with BPH than in controls (7.0% vs. 2.8%, odds ratio = 2.59, 95% confidence interval, 0.94-7.13, not statistically significant). We concluded that germline/somatic mutations of HPC2/ELAC2 are uncommon in PCa. Similarly, allelic imbalances at the gene locus and changes in expression are rare. Although no difference in allele frequency at Ser217Leu between patients with PCa and controls has been reported in a Western population, this polymorphism is a potential indicator of PCa risk in Japanese men and it should be examined in other ethnic groups.

Estrous cyclicity and ovarian follicles in female rats after prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl.

Alterations of estrous cyclicity and ovarian follicles following prenatal exposure to PCB126 were examined. Female SD rats were given (i.g.) 25 pg, 2.5, 250 ng and 7.5 microg of PCB126/kg or the vehicle on days 13-19 postconception. Vaginal opening (VO) in the 250 ng and 7.5 microg offspring was significantly delayed. All groups showed irregular estrous cyclicity following VO, but it became normal after a few days. However, the start of normal estrous cyclicity following VO in the 2.5, 250 ng and 7.5 microg groups was significantly delayed. At 30 and 50 days old, the 2.5, 250 ng and 7.5 microg groups showed significantly fewer antral follicles and a higher number of atretic follicles. The 7.5 microg group at 50 days old revealed significantly fewer corpus luteums. In 50-day-old offspring, the 2.5, 250 ng and 7.5 microg groups showed a significant reduction in serum 17beta-estradiol and progesterone levels and significantly higher levels of PCB126 in the fatty tissue compared with the vehicle group. Thus, while the prenatal dose of PCB126 used in this study did not induce malformation of the external genitalia or persistent ovarian disruption, disruption of ovarian function at puberty was found in the 2.5 ng group of pups born to dams exposed to 17.5 ng/kg PCB126. The present study suggests that PCB126, at least in part, exerted direct effects on the ovary as shown by the disruption of estrous cyclicity.

Thirteen-week repeated dose toxicity study of wormwood (Artemisia absinthium) extract in rats.

Wormwood, Artemisia absinthium, is a very bitter plant, and its extract has been used as food additives such as seasonings for food and drinks. A 13-week repeated dose toxicity study of wormwood extract was performed in both sexes of Wistar Hannover (GALAS) rats. Rats were divided into 4 groups consisting of 10 males and 10 females each, and were given water containing 0, 0.125, 0.5, or 2% wormwood extract. All rats had survived at the end of the study, and no changes indicating obvious toxicities that are attributable to the treatment of wormwood extract were observed in the body weights, hematological and serum biochemical examinations, organ weights, and histopathological examinations. Based on the results of the present study, the NOAEL (no-observed-adverse-effect-level) of wormwood extract of Wistar Hannover rats was estimated to be 2% (equivalent to 1.27 g/kg/day in males and 2.06 g/kg/day in females) or more.

Mammary gland differentiation in female rats after prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl.

Recently we reported finding that prenatal exposure to a relatively low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) increases the rate of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreases it. One of the most important factors determining sensitivity of the mammary gland to neoplastic stimuli is its stage of differentiation at the time of exposure to the carcinogenic agent. Hence, to verify a biphasic dose-response relationship (enhancement of carcinogenesis at low dose, and inhibition at high dose), we investigated the effects of prenatal exposure to PCB126 on mammary gland differentiation. Female SD rats were injected (i.g.) with 25 pg, 2.5 ng, 250 ng, 7.5 microg of PCB126/kg, or the vehicle, on days 13-19 postconception. In 50-day-old offspring, regardless of the day of exposure to DMBA, only the 7.5 microg group showed statistically significant high levels of PCB126 in the fatty tissue of their mammary glands. Fifty-day-old female offspring of the 250 ng group showed apparent inhibition of the normal differentiation of terminal end buds (TEB) to alveolar buds and lobules (ABL), while those of the 7.5 microg group showed mammary gland hypoplasia. Expression levels of the estrogen receptor-alpha (ER) in TEBs and the ER mRNA in mammary glands were higher in the 7.5 microg, 250 ng, 2.5 ng groups. Proliferating cell nuclear antigen (PCNA) expression in TEBs of 50-day-old rats was statistically significantly higher in the 250 ng group and lower in the 7.5 microg group. In the developing mammary gland, TEBs are considered the most susceptible to mammary carcinogenesis, while ABLs are relatively protected from mammary carcinogenesis. Thus, prenatal exposure to a relatively low dose of PCB126 induced an alteration of mammary gland differentiation that might potentially increase the risk of DMBA-induced mammary carcinoma.