Developmental effects of estrogen and PCBs in the reproductive tract of BALB/C mice

Many of the tumorigenic effects that result from neonatal exposure to both natural and synthetic estrogens resemble those found in humans exposed to diethylstilbestrol (DES) in utero. Using this established DES neonatal mouse model, my goal was to investigate long-term molecular and morphological effects of certain polychlorinated biphenyls (PCBs) that are weakly estrogenic in adult mice. Focusing on the cervicovaginal (CV) tract, since this is where tumors develop in the BALB/c mouse, I first assessed the 17β-estradiol (E2) dose-response for expression of lactoferrin (LTF). LTF is a highly inducible estrogen biomarker that is permanently altered in uteri from neonatally treated mice. Treatments were administered via 5 subcutaneous injections beginning within 16 hrs after birth, days 1–5. ^ The ontogeny of LTF expression from mouse CV tracts was determined by examining three different stages of life: pups, immature, and mature mice. Northern RNA analysis and immunohistochemistry showed that neonatal E 2 treatment both increases and decreases LTF expression. Early expression of LTF in the CV tract at all doses occurred in pups. In both immature and adult mice, increased LTF expression was dependent on whether E2 induced ovary-dependent or ovary-independent persistent vaginal cornification. ^ Next, I studied biological responses from neonatally PCB exposed adult mice. As expected, using a neonatal uterine bioassay I showed that 2 ′4′6′-trichloro-4-biphenylol (OH-PCB-30), 2′3′4′ 5-tetrachloro-4-biphenyloI (OH-PCB-61), and OH-PCB-30/61 (50/50 mixture), were estrogenic causing a dose-dependent increase in uterine weight. ^ Long-term effects of OH-PCB 30 [200 μg/pup/day] were most similar to E2 as seen by an increased uterine wet weight in day 50 mice similar to E2 [5 μg/pup/day] (141% and 140% of control, respectively). Another similarity between OH-PCB 30 and E2 neonatally treated mice was found in those sacrificed at 20 months of age. At these same doses CV tract squamous cell carcinoma induction was 43% of E2 treated mice and 47% of OH-PCB 30 treated mice. Differences were noted in adenoaquamous; cell carcinoma development, where 16% of OH-PCB-30 neonatally treated mice developed tumors versus 8% for E2. Based on these results using the neonatal mouse model, I conclude that the OH-PCBs tested are strongly estrogenic and tumorigenic showing dose-response relationships when exposure occurs during development of the reproductive tract in mice. These results may have important implications for risk assessment in determining the effects of xenoestrogens exposure early versus later in life. ^

The role of chronic irritation and inflammation in tumor promotion in CD-1 mice by petroleum middle distillates

An initiation-promotion bioassay in CD-1 mice was used to examine the role of chronic irritation and inflammation in tumor promotion by petroleum middle distillates. A representative hydrodesulfurized middle distillate (API 81-07) was selected as the test article. Test groups (54 mice per group) were initiated once with 50 ug of 7,12-dimethylbenzanthracene (DMBA). Promotion with API 81-07 consisted of twice weekly treatments for 25 weeks with either 25 ul, 50 ul, 50 ul + daily treatment with 15 ug dexamethasone, 50 ul + post-application washings, and 100 ul. Three mice from each group were sacrificed at 21 day intervals (24 total per group). The skin from interim sacrificed (IS) mice was examined histopathologically for tumor, acanthosis, hyperkeratosis, pseudo-epitheliomatous hyperplasia, epidermal crusting, and subacute inflammation. In-life observations included examination of all mice for erythema and edema for 8 weeks following the first promotion treatment. Tumor incident at study termination was as follows: 25 ul (45%), 50 ul (43%), 50 ul + dexamethasone (0%), 50 ul + washing (70%), and 100 ul (81%). An overall correlation of $>$0.90 between tumor incidence and group means for acanthosis and hyperkeratosis was observed in IS mice at all intervals. Correlations of $<$0.75 were observed for other group mean histopathological parameters and tumor incidence. The overall correlation of group mean erythema and edema with tumor incident was $>$0.90. The results of this study support the hypothesis that induction of a lasting, albeit mild, hyperplasia is an essential, but not sufficient requirement, for tumor promotion. Furthermore, subacute inflammation does not appear to be a significant factor in tumor promotion by petroleum middle distillates. However, inflammation may be a factor in tumor progression. ^

Design of long-term animal bioassay for low-dose extrapolation using a multistage model

Conventional designs of animal bioassays allocate the same number of animals into control and dose groups to explore the spontaneous and induced tumor incidence rates, respectively. The purpose of such bioassays are (a) to determine whether or not the substance exhibits carcinogenic properties, and (b) if so, to estimate the human response at relatively low doses. In this study, it has been found that the optimal allocation to the experimental groups which, in some sense, minimize the error of the estimated response for low dose extrapolation is associated with the dose level and tumor risk. The number of dose levels has been investigated at the affordable experimental cost. The pattern of the administered dose, 1 MTD, 1/2 MTD, 1/4 MTD,....., etc. plus control, gives the most reasonable arrangement for the low dose extrapolation purpose. The arrangement of five dose groups may make the highest dose trivial. A four-dose design can circumvent this problem and has also one degree of freedom for testing the goodness-of-fit of the response model.^ An example using the data on liver tumors induced in mice in a lifetime study of feeding dieldrin (Walker et al., 1973) is implemented with the methodology. The results are compared with conclusions drawn from other studies. ^

TEMPORAL VARIATION OF ACUTE AND LUNG TUMORIGENIC EFFECTS OF URETHAN ON STRAIN A MICE (CHRONOBIOLOGY, CARCINOGENICITY, ANESTHETIC, DNA SYNTHESIS)

The effect of circadian variation on susceptibility to the chemical induction of cancer was assessed utilizing the mouse pulmonary adenoma bioassay. Different groups of male A/Jax mice (standardized for rhythm analysis with light from 0600-1800 and darkness from 1800-0600) each received a single timed i.p. injection of urethan (Bioassay I: 0.25, 0.5 or 1.0 mg/g body weight; Bioassay II: 0.75, 1.0, 1.25 mg/g body weight; Bioassay III: 1.0 mg/g body weight) at the following times, 0100, 0500, 0900, 1300, 1700 or 2100. Mice were sacrificed 16 weeks after treatment. The tumorigenic effect of urethan on the lungs (lung surface pulmonary adenomas) was assessed. In addition, mortality, body weight changes and the anesthetic effect of urethan were determined. The rhythmic pattern of DNA synthesis in the lung and the comparative rhythmic pattern in the liver were assessed using a tritiated thymidine incorporation assay.^ In the first adenoma bioassay, the lung tumorigenic response in mice given the highest dose of urethan exhibited a 12-hour rhythm with a major peak in tumor yield at 0100 and a secondary peak at 1300; reduced yields occurred at 0500-0900 and 2100. The second adenoma bioassay, studied at a 6-month seasonal divergence in time from the first study showed a peak at 1300 but not at 0100. The mice from the third adenoma bioassay, studied at an 11-month seasonal divergence in time from the 2nd study showed an increase in tumor yield during the rest cycle (0900-1700).^ This study found a definite suggestion of a low amplitude rhythm in susceptibility to urethan induced effects. The acute toxic and pharmacological effects correlated to exhibit a maximal effect during dark hours (activity span). This rhythmicity might be explained by an alteration in the amplitude of hepatic metabolism. The chronic carcinogenic response exhibited an opposite pattern. Urethan induced tumor response was greater during daylight hours (rest cycle). This correlated with the slight elevation in DNA synthetic activity found in the lung and liver which might be responsible for the increase in carcinogenic response. (Abstract shortened with permission of author.) ^