Sherry M. Lewis

Toxicity Evaluation of Bisphenol A Administered by Gavage to Sprague Dawley Rats From Gestation Day 6 Through Postnatal Day 90

Bisphenol A (BPA) is a high production volume industrial chemical to which there is widespread human oral exposure. Guideline studies used to set regulatory limits detected adverse effects only at doses well above human exposures and established a no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day. However, many reported animal studies link BPA to potentially adverse effects on multiple organ systems at doses below the NOAEL. The primary goals of the subchronic study reported here were to identify adverse effects induced by orally (gavage) administered BPA below the NOAEL, to characterize the dose response for such effects and to determine doses for a subsequent chronic study. Sprague Dawley rat dams were dosed daily from gestation day 6 until the start of labor, and their pups were directly dosed from day 1 after birth to termination. The primary focus was on seven equally spaced BPA doses (2.5-2700 μg/kg bw/day). Also included were a naïve control, two doses of ethinyl estradiol (EE2) to demonstrate the estrogen responsiveness of the animal model, and two high BPA doses (100,000 and 300,000 μg/kg bw/day) expected from guideline studies to produce adverse effects. Clear adverse effects of BPA, including depressed gestational and postnatal body weight gain, effects on the ovary (increased cystic follicles, depleted corpora lutea, and antral follicles), and serum hormones (increased serum estradiol and prolactin and decreased progesterone), were observed only at the two high doses of BPA. BPA-induced effects partially overlapped those induced by EE2, consistent with the known weak estrogenic activity of BPA.

Review of Usnic Acid and Usnea Barbata Toxicity

Usnic acid is a prominent secondary lichen metabolite that has been used for various purposes worldwide. Crude extracts of usnic acid or pure usnic acid have been marketed in the United States as dietary supplements to aid in weight loss. The US Food and Drug Administration (FDA) received 21 reports of liver toxicity related to the ingestion of dietary supplements that contain usnic acid. This prompted the FDA to issue a warning about one such supplement, LipoKinetix, in 2001 (http://www.cfsan.fda.gov/~dms/ds-lipo.html). Subsequently, usnic acid and Usnea barbata lichen were nominated by the National Toxicology Program (NTP) for toxicity evaluations. At present, a toxicological evaluation of usnic acid is being conducted by the NTP. This review focuses on the recent findings of usnic acid-induced toxicities and their underlying mechanisms of action.

Oxidative DNA damage levels in rats fed low-fat, high-fat, or calorie-restricted diets

Increased fat and caloric content of the diet has been associated with increased mammary tumor incidence. The dietary modulation of cellular redox state may be one mechanism behind this association. We have examined the effects of changes in dietary fat and caloric intake on the levels of 5-hydroxymethyluracil in DNA from rat liver and mammary gland. Female Fischer 344 rats, 40 days old, were maintained on 3% (low-fat), 5% (control), or 20% (high-fat) corn oil diets for 2 weeks. A fourth group of rats had the same daily fat intake as the control group, but total caloric intake was restricted by 40%. As a measure of oxidative DNA damage, 5-hydroxymethyluracil levels were measured in the DNA extracted from liver and mammary gland by gas chromatography-mass spectrometry. 5-Hydroxymethyluracil levels in the liver DNA of the low-fat, high-fat, and calorie-restricted groups were decreased relative to that of control, but the only significant decrease was in the calorie-restricted group (p less than 0.01). In the mammary gland DNA, statistically significant decreases in damage were found in each group relative to control (p less than 0.05). The relationship between fat in the diet and oxidative stress is thus complex. These results show that changes in dietary intake of both fat and calories can modulate oxidative DNA damage levels, and the effect of diet was more clearly evident in the DNA from mammary gland than in DNA from liver.

The effects of different levels of dietary restriction on aging and survival in the Sprague-Dawley rat: Implications for chronic studies

A study was undertaken to determine the effects of incremental levels of dietary restriction (DR) in rats. Survival, growth, reproductive, and dietary intake (DI) variables were monitored in a chronic study in which male Sprague Dawley (SD) rats (NCTR colony) were fed their ration ad libitum (AL), or DR. The main objectives were to determine if low levels of DR could be used to increase the survival rate of SD rats in the chronic bioassay, and to identify the survival characteristics of a long-lived SD rat strain (NCTR colony). The average life span of AL rats was 115 months. At 104 weeks on study (110 weeks of age), the survival rate for the AL and 10%, 25%, and 40% DR groups was 63.4, 87.5, 87.5, and 97.5%, respectively. The largest increase in survival (24.1%) occurred between AL and 10% DR, indicating that very low levels of DR have a significant effect on survival. Whole-body, liver, prostate, and epididymis weights and body length were decreased by DR, whereas brain weight, testicular weight, and skull length were not altered by DR. Rats from the NCTR colony were found to be ideal for chronic studies because they are much longer-lived than other SD stocks. Although the 104-week survival rate for these SD, non-obese AL rats exceeds the FDA’s “Redbook” survival guideline (> 50%) for chronic bioassays, the use of DR is advocated because it reduces individual variability in body weight.

Investigation of the Effects of Subchronic Low Dose Oral Exposure to Bisphenol A (BPA) and Ethinyl Estradiol (EE) on Estrogen Receptor Expression in the Juvenile and Adult Female Rat Hypothalamus

Concerns have been raised regarding the long-term impacts of early life exposure to the ubiquitous environmental contaminant bisphenol A (BPA) on brain organization. Because BPA has been reported to affect estrogen signaling, and steroid hormones play a critical role in brain sexual differentiation, there is also concern that BPA exposure could alter neural sex differences. Here, we examine the impact of subchronic exposure from gestation to adulthood to oral doses of BPA below the current no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day on estrogen receptor (ESR) expression in sexually dimorphic brain regions of prepubertal and adult female rats. The dams were gavaged daily with vehicle (0.3% carboxymethylcellulose), 2.5, 25, 260, or 2700 μg BPA/kg bw/day, or 0.5 or 5.0 μg ethinyl estradiol (EE)/kg bw/day from gestational day 6 until labor began. Offspring were then gavaged directly from the day after birth until the day before scheduled sacrifice on postnatal days 21 or 90. Using in situ hybridization, one or more BPA doses produced significant decreases in Esr1 expression in the juvenile female rat anteroventral periventricular nucleus (AVPV) of the hypothalamus and significant decreases in Esr2 expression in the adult female rat AVPV and medial preoptic area (MPOA), relative to vehicle controls. BPA did not simply reproduce EE effects, indicating that BPA is not acting solely as an estrogen mimic. The possible consequences of long-term changes in hypothalamic ESR expression resulting from subchronic low dose BPA exposure on neuroendocrine effects are discussed and being addressed in ongoing, related work.

Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice.

Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F(1) female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I-IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.

Effect of varying dietary fat levels on rat growth and oxidative DNA damage.

Dietary fat has previously been shown to have somewhat complicated relationships to levels of oxidative stress in rats. In this study, we examined the effects of five different dietary fat intakes on levels of oxidative DNA damage in rats. Animals fed diets containing 3%, 5%, 10%, or 15% corn oil had body weights that were similar after 20 weeks. Animals fed a 20% fat diet, however, had significantly higher mean body weight than any other group. Levels of 5-hydroxymethyl-2′-deoxyuridine, one marker of oxidative DNA damage, had different relationships to dietary fat in blood and mammary gland. In blood, levels increased with dietary fat levels, and the highest levels were observed with the 20% fat diet (65% higher levels than with the 3% fat diet). In mammary gland, a plateau-type effect was observed, with maximal levels of oxidative DNA damage being obtained using 10% fat (representing a 68% increase relative to the 3% fat diet). This could be a result of induction of compensatory mechanisms in response to a high-fat diet in mammary gland but not in the short-lived nucleated blood cells. Oxidative DNA damage levels in blood thus appear to be a marker of dietary fat intake. In mammary gland, however, levels of DNA damage are consistent with previously observed promotional effects of dietary fat on mammary gland tumorigenesis at lower levels of fat intake with little or no incremental promoting effects at higher levels of fat intake.

Effects of developmental exposure to bisphenol A on spatial navigational learning and memory in rats: A CLARITY-BPA study.

Bisphenol A (BPA) is a ubiquitous industrial chemical used in the production of a wide variety of items. Previous studies suggest BPA exposure may result in neuro-disruptive effects; however, data are inconsistent across animal and human studies. As part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), we sought to determine whether female and male rats developmentally exposed to BPA demonstrated later spatial navigational learning and memory deficits. Pregnant NCTR Sprague-Dawley rats were orally dosed from gestational day 6 to parturition, and offspring were directly orally dosed until weaning (postnatal day 21). Treatment groups included a vehicle control, three BPA doses (2.5μg/kg body weight (bw)/day-[2.5], 25μg/kg bw/day-[25], and 2500μg/kg bw/day-[2500]) and a 0.5μg/kg/day ethinyl estradiol (EE)-reference estrogen dose. At adulthood, 1/sex/litter was tested for seven days in the Barnes maze. The 2500 BPA group sniffed more incorrect holes on day 7 than those in the control, 2.5 BPA, and EE groups. The 2500 BPA females were less likely than control females to locate the escape box in the allotted time (p value=0.04). Although 2.5 BPA females exhibited a prolonged latency, the effect did not reach significance (p value=0.06), whereas 2.5 BPA males showed improved latency compared to control males (p value=0.04), although the significance of this result is uncertain. No differences in serum testosterone concentration were detected in any male or female treatment groups. Current findings suggest developmental exposure of rats to BPA may disrupt aspects of spatial navigational learning and memory.

Nutrient intake and growth characteristics of male Sprague-Dawley rats fed AIN-93M purified diet or NIH- 31 natural-ingredient diet in a chronic two-year study

Background and aims: Daily nutrient intake and growth of male Sprague-Dawley (SD) rats fed compositionally different diets were monitored over 114 weeks to determine whether rats fed ad libitum (AL) or diet restricted (DR) followed normal growth parameters. A second objective was to evaluate the usefulness of the American Institutes of Nutrition’s AIN-93M (maintenance formulation) diet for aging and DR studies. Methods: Rats were fed NIH-31 cereal-based diet AL, or a vitamin-fortified modification of the NIH-31 diet at 10, 25, or 40% DR. Other SD rats were fed AIN-93M diet AL or 31% DR; daily nutrient intake and growth response were reported. Results: At all intervals up to 36 weeks of age, rats fed AL the NIH-31 diet consumed significantly (p<0.001) more than rats fed AL the AIN-93M diet, and required more diet per unit of gain than AIN-93M AL rats. However, body weight (BW) gain in rats AL-fed the AIN-93M diet demonstrated that energy components were more efficiently metabolized than in those fed the NIH-31 diet. Whereas diet restriction decreased BW, the rate of maturation, i.e., the rate of reaching a mature BW, increased as intake level decreased. Growth response showed all growth curves were normal, but intake level effects on mature BW and maturation rate differed significantly (p<0.001). Curves for rats AL- and DR-fed the AIN-93M diets were similar to those of rats AL- and DR-fed NIH-31 diet formulations, suggesting that diets adequately met growth requirements and supported normal growth parameters of male SD rats when fed AL or DR. Conclusions: A modification in the AIN-93M energy components to reduce total calories and an evaluation of other nutrient profiles could improve its usefulness as a maintenance and aging diet.

The effects of different levels of dietary restriction on non-neoplastic diseases in male Sprague-Dawley rats

Background and aims: The primary purpose of the present study was to investigate the effects of 10, 25, and 40% dietary restriction (DR) on non-neoplastic diseases in rodents at 58 and 110 weeks of age, and to determine whether low-level DR (10 and 25%) can increase the survival rate and decrease variability in chronic bioassay studies. Methods: Male Sprague-Dawley (SD) rats (NCTR colony) were divided into four nutritional groups, consisting of an ad libitum (AL) group with unlimited access to the NIH-31 diet, and three dietary restricted (DR) groups given the NIH-31 diet reduced in amount by 10, 25, and 40%. Results: At 110 weeks of age, the incidence of cardiomyopathy was 95, 75, 45, and 15% for AL and 10, 25, and 40% DR rats, respectively; the incidence of nephropathy was 55, 20, 15, and 0% for AL and 10, 25, and 40% DR rats, respectively. The severity of chronic heart and kidney diseases was significantly reduced in all DR rat groups, with significant DR-dependent linear trends for these diseases. Moreover, DR prevented the progression of skin irritation to foot ulcers, and reduced the age-related degeneration in the adrenal, lacrimal, and thymus glands, and the liver. Conclusions: These results clearly indicate that even low DR levels were effective in preventing or slowing the progression of these non-neoplastic diseases.