Photo-co-carcinogenesis of Topically Applied Retinyl Palmitate in SKH-1 Hairless Mice.

Cosmetic products that contain retinyl palmitate are popular as antiaging skin treatments; however, recent studies suggest a risk for enhanced skin tumor development with topical retinyl palmitate applications and exposure to solar ultraviolet radiation (UVR). In this study, we investigated the potential of retinyl palmitate to enhance UVR-induced photo-co-carcinogenesis. Groups of 36 male and 36 female SKH-1 hairless mice were exposed to simulated solar light (SSL) and treated with the control cream or creams containing retinyl palmitate, 5 days per week for 40 weeks. Other groups of mice were exposed to SSL and received no cream treatment or received cream treatments and were exposed to ultraviolet-A or ultraviolet-B. Mice were monitored for the development of skin tumors, and the incidences and multiplicities of squamous cell neoplasia were determined by histopathology. In both the absence and presence of SSL, mice administered the control cream developed skin tumors earlier and had higher incidences and multiplicities of skin squamous cell neoplasms than mice that received no cream treatment. Compared to the control cream groups, mice exposed to SSL and administered the retinyl palmitate creams demonstrated earlier onsets of skin tumors and had increased incidences and multiplicities of squamous cell skin neoplasms.

Low dose assessment of the carcinogenicity of furan in male F344/N Nctr rats in a 2-year gavage study.

Furan is a volatile organic chemical that is a contaminant in many common foods. Furan is hepatocarcinogenic in mice and rats; however, the risk to humans from dietary exposure to furan cannot be estimated accurately because the lowest tested dose of furan in a 2-year bioassay in rats gave nearly a 100% incidence of cholangiocarcinoma. To provide bioassay data that can be used in preparing risk assessments, the carcinogenicity of furan was determined in male F344/N Nctr rats administered 0, 0.02, 0.044, 0.092, 0.2, 0.44, 0.92, and 2 mg furan/kg body weight (BW) by gavage 5 days/week for 2 years. Exposure to furan was associated with the development of malignant mesothelioma on membranes surrounding the epididymis and on the testicular tunics, with the increase being significant at 2 mg furan/kg BW. There was also a dose-related increase in the incidence of mononuclear cell leukemia, with the increase in incidence being significant at 0.092, 0.2, 0.92, and 2 mg furan/kg BW. Dose-related non-neoplastic liver lesions included cholangiofibrosis, mixed cell foci, basophilic foci, biliary tract hyperplasia, oval cell hyperplasia, regenerative hyperplasia, and cytoplasmic vacuolization. The most sensitive non-neoplastic lesion was cholangiofibrosis, the frequency of which increased significantly at 0.2 mg furan/kg BW.

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.

Clear evidence of carcinogenic activity by a whole-leaf extract of Aloe barbadensis miller (aloe vera) in F344/N rats.

Aloe barbadensis Miller (Aloe vera) is an herbal remedy promoted to treat a variety of illnesses; however, only limited data are available on the safety of this dietary supplement. Drinking water exposure of F344/N rats and B6C3F1 mice to an Aloe vera whole-leaf extract (1, 2, and 3%) for 13 weeks resulted in goblet cell hyperplasia of the large intestine in both species. Based upon this observation, 2-year drinking water studies were conducted to assess the carcinogenic potential of an Aloe vera whole-leaf extract when administered to F344/N rats (48 per sex per group) at 0.5, 1, and 1.5%, and B6C3F1 mice (48 per sex per group) at 1, 2, and 3%. Compared with controls, survival was decreased in the 1.5% dose group of female rats. Treatment-related neoplasms and nonneoplastic lesions in both species were confined primarily to the large intestine. Incidences of adenomas and/or carcinomas of the ileo-cecal and cecal-colic junction, cecum, and ascending and transverse colon were significantly higher than controls in male and female rats in the 1 and 1.5% dose groups. There were no neoplasms of the large intestine in mice or in the 0 or 0.5% dose groups of rats. Increased incidences of mucosa hyperplasia of the large intestine were observed in F344/N rats, and increased incidences of goblet cell hyperplasia of the large intestine occurred in B6C3F1 mice. These results indicate that Aloe vera whole-leaf extract is an intestinal irritant in F344/N rats and B6C3F1 mice and a carcinogen of the large intestine in F344/N rats.

Genistein and ethinyl estradiol dietary exposure in multigenerational and chronic studies induce similar proliferative lesions in mammary gland of male Sprague-Dawley rats.

Genistein and ethinyl estradiol (EE(2)) were examined in multigenerational reproductive and 2-yr chronic toxicity studies with different exposure durations across generations F(0) through F(4). Sprague-Dawley rats were exposed to genistein (0, 5, 100, or 500 ppm) or EE(2) (0, 2, 10, or 50 ppb). Effects in the male mammary gland are described here. In the multigeneration studies, mammary hyperplasia was induced by both compounds; the chronic studies had a lower incidence, without proportionate neoplasia. Sexual dimorphism (predominant tubuloalveolar growth in females and lobuloalveolar in males) was retained without feminization in high dose genistein or EE(2). In the continuously exposed generations, mammary hyperplasia was sustained but not amplified, appeared morphologically similar across all generations, and was not carried over into unexposed offspring of previously exposed generations. The hyperplasia in male rats was similar whether induced by genistein or EE(2). Results substantiate and extend previous reports that mammary gland hyperplasia in the male rat is one of the most sensitive markers of estrogenic endocrine disruption.

Overlapping but distinct effects of genistein and ethinyl estradiol (EE(2)) in female Sprague-Dawley rats in multigenerational reproductive and chronic toxicity studies.

Genistein and ethinyl estradiol (EE(2)) were examined in multigenerational reproductive and chronic toxicity studies that had different treatment intervals among generations. Sprague-Dawley rats received genistein (0, 5, 100, or 500 ppm) or EE(2) (0, 2, 10, or 50 ppb) in a low phytoestrogen diet. Nonneoplastic effects in females are summarized here. Genistein at 500 ppm and EE(2) at 50 ppb produced similar effects in continuously exposed rats, including decreased body weights, accelerated vaginal opening, and altered estrous cycles in young animals. At the high dose, anogenital distance was subtly affected by both compounds, and a reduction in litter size was evident in genistein-treated animals. Genistein at 500 ppm induced an early onset of aberrant cycles relative to controls in the chronic studies. EE(2) significantly increased the incidence of uterine lesions (atypical focal hyperplasia and squamous metaplasia). These compound-specific effects appeared to be enhanced in the offspring of prior exposed generations.

Neoplastic pathology in male Sprague-Dawley rats fed AIN-93M diet ad libitum or at restricted intakes.

The primary purpose of this study was to evaluate the effects of age and long-term dietary reduction on neoplastic diseases in rats fed the AIN-93M purified diet. Second, pathologic profiles are critical to comprehensive dietary evaluation. Male Sprague-Dawley rats assigned to 2 groups, ad libitum (AL) and dietary restricted (DR), were fed the AIN-93M (casein protein) diet free choice and reduced in amount by 31%, respectively. At 58 weeks of age, the predominant types of lesions in AL and DR rats were pituitary and skin tumors. At 114 weeks of age, the most common lesions were pituitary, adrenal gland, skin, mammary, brain, and pancreatic tumors and mononuclear cell leukemia. However, DR had no significant effect on these lesions. Primary findings demonstrate that DR significantly reduced the total number of tumors per rat and incidence of benign and primary tumors (all organs) but did not reduce the incidence of malignant tumors (all organs). Dietary restriction increased the percentage of unknown deaths. These results may explain why survival rates for AL and DR rats were not significantly different at 114 weeks (43.3 vs 57.5%, respectively). These findings differ from previous studies using NIH-31 cereal diet (Aging Clin Exp Res 2001;13:263; J Nutr 2002;132:101; Aging Clin Exp Res 2003;16(6):68; Aging Clin Exp Res 2004;16:448) where neoplastic lesions rather than nonneoplastic lesions were linked to a significant increase in survival rate among cohorts of DR-fed rats (J Nutr 2002;132:101). Factors such as diet composition and digestibility, although not independent of body weight, may have contributed to differences in rat mortality and may affect humans in a similar manner.

Nonneoplastic pathology in male Sprague-Dawley rats fed the American Institute of Nutrition-93M purified diet at ad libitum and dietary-restricted intakes.

This study evaluates the effects of age and chronic dietary restriction (DR) on nonneoplastic diseases in rats that were fed the American Institute of Nutrition (AIN)-93M purified diet. Male Sprague-Dawley (SD) rats were divided into an ad libitum (AL) group and a DR group that was fed the AIN-93M diet with intake reduced by 31%. Nonneoplastic disease profiles were developed to clarify whether the AIN-93M diet fulfills long-term nutritional requirements of rats. Subsets of rats were killed at 58 and 114 weeks of age, and histopathology was performed. At 58 weeks of age, the 2 main types of nonneoplastic diseases in AL rats were liver vacuolization and cardiomyopathy. Dietary restriction reduced the severity and incidence of both lesions. At 114 weeks of age, the most common lesions in AL rats were cardiomyopathy, nephropathy, liver vacuolization, and degeneration with renal failure and genitourinary infections causing the greatest mortality. Dietary restriction reduced the incidence and severity of these lesions. Nonneoplastic diseases accounted for 28.9% and 0.0% of total mortalities in the AL and DR groups, respectively; however, there was a higher incidence of unknown deaths in the DR rats (52.6%) compared to AL rats (28.9%), which may have limited the success of DR to improve survival. Although the AIN-93M diet supported chronic rat growth, alterations in some dietary component concentrations may be required to lower body weight in chronic rodent and human studies. Factors such as diet composition and digestibility may alter nonneoplastic diseases and mortality in rats and humans in a similar fashion.

The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies.

BACKGROUND: Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists. RESULTS: Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan - the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent P-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on P-value ranking is an expected mathematical consequence of the high variability of the t-values; the more stringent the P-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations. CONCLUSION: We recommend the use of FC-ranking plus a non-stringent P cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the P-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and P-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the P criterion balances sensitivity and specificity.

Identification of rat hippocampal mRNAs altered by the mitochondrial toxicant, 3-NPA.

3-Nitropropionic acid (3-NPA) is a model mitochondrial inhibitor that causes selective neurodegeneration in brain. 3-NPA-induced neurodegeneration occurs via a secondary neurotoxicity, caused initially by ATP depletion and redox changes in the cell. It is known that the hippocampal degeneration caused by mitochondrial dysfunction affects learning and memory, cognitive functions commonly disturbed in neurodegenerative diseases. The 3-NPA- treated animal model can be used to study molecular mechanisms underlying selective degeneration in the brain. In this study, a microarray approach was utilized to define changes in the expression of 530 genes in the rat hippocampus after acute exposure to 3-NPA at 30 mg/kg, sc. The microarray data were collected at 30 min, 2 h, and 4 h post-3-NPA. Statistical modeling using an ANOVA mixed model applied to Van der Waerden scores of rank-transformed intensity data was used to assign statistical significance to 44 transcripts. These transcripts represent genes associated with energy metabolism, calcium homeostasis, the cytoskeleton, neurotransmitter metabolism, and other cellular functions. Changes in the transcripts of genes encoding 2 transporters [blood-brain specific anion transporter (Slco1c1) and sodium-dependent inorganic phosphate cotransporter (Slc17a7)] were confirmed by real-time RT-PCR. In conclusion, this study identified 2 new potential targets for enhancement of neuroprotection or inhibition of neurodegeneration associated with ATP depletion in the hippocampus.

Inhibition of platelet aggregation with eptifibatide, bivalirudin, and heparin in patients undergoing percutaneous coronary intervention receiving clopidogrel pretreatment (The PharmacoDynamic Evaluation of Angiomax, Clopidogrel with or without INtegrilin [DEACON] study).

The present study assessed the magnitude of inhibition of platelet aggregation induced by adenosine diphosphate and thrombin receptor activating peptide achieved with unfractionated heparin, the direct thrombin inhibitor bivalirudin, and the glycoprotein IIb/IIIa inhibitor eptifibatide in patients who underwent percutaneous coronary intervention and and were treated with concomitant aspirin and clopidogrel.

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.

Bradycardia during therapy for multiple myeloma with thalidomide.

We studied the medical records of 96 patients who received thalidomide and 104 patients who made up a control group. We found that 53% of patients (52 patients) using thalidomide had a heart rate of <60 beats/min at some point during follow-up and 19% of thalidomide patients (10 patients) developed symptom-related bradycardia. Reducing the thalidomide dose appeared to alleviate symptoms in most patients.

Electroencephalographic, behavioral, and c-fos responses to acute domoic acid exposure.

Domoic acid, a potent excitotoxic analogue of glutamate and kainate, may cause seizures, amnesia, and sometimes death in humans consuming contaminated shellfish. Continuous behavioral observations and recordings of the electrocorticogram (ECoG, via bipolar, epidural electrodes) were obtained from nonanesthetized rats for 2 h after intraperitoneal injection with either saline, 2.2, or 4.4 mg/kg of domoic acid. Rats were then sacrificed for c-fos immunohistochemistry. Fast Fourier transformation (FFT) of the ECoG data to obtain the voltage as a function of frequency indicated that the lower frequency bands (theta, 4.75-6.75 Hz and delta, 1.25-4.50 Hz) were the first to respond, with a significant elevation by 30 min after the high dose of domoic acid. The lower dose of domoic acid also caused a significant elevation of ECoG voltage, but not until later in the session. Sixty minutes after dosing, the behavioral biomarkers of "ear scratching" and "rearing, praying" (RP) seizures became significantly elevated in the high-dose rats. The low-dose rats showed no significant alterations in behavior at any time during the session. In postmortem brains obtained immediately after the sessions, c-fos was activated in the anterior olfactory nucleus by both the low and high doses of domoic acid. However, only the high dose increased c-fos immunoreactivity in the hippocampus, affecting both the granule and pyramidal neurons. These data indicate that electroencephalographic and c-fos responses can be obtained at a dose of domoic acid that fails to activate the behavioral response most commonly used as a bioassay for this marine toxin: ear scratching with the ipsilateral foot.

The effects of different levels of dietary restriction on neoplastic pathology in the male Sprague-Dawley rat.

BACKGROUND AND AIMS: The primary purpose of this study was to evaluate the effects of varied levels of dietary restriction (DR) on neoplastic pathologies in rodents at 58 and 110 weeks of age. METHODS: Male Sprague-Dawley (SD) rats were divided into four nutritional groups; an ad libitum (AL) control group, and three dietary restricted (DR) groups that were fed the NIH-31 diet reduced in amount by 10, 25, and 40%. RESULTS: At 110 weeks of age, compared to AL rats, the incidence of benign tumors was significantly lower in all DR groups while primary tumors were significantly lower in the 10 and 40% DR groups; no malignant tumors were detected in the 10% DR group. Most defined mortalities were caused by neoplastic lesions. All levels of DR reduced the percentage of tumor-bearing animals, the incidence of skin tumors (combined), and the total number of tumors. Pituitary, skin, and pancreatic tumors were the most prolific lesions; pituitary and skin tumors were the most fatal. Compared to AL rats, the time to onset of skin and pancreatic tumors was longer in all of the DR groups. CONCLUSION: In many cases, the incidences of neoplastic lesions were similar among the DR groups, clearly indicating that the DR effect is not linear and that even a very low level of DR (10%) can have a significant effect on many important neoplastic lesions and tumor burden. The main effect of DR was to decrease the incidence of some neoplastic lesions and to increase the time to onset and/or decrease the progression of tumors, thereby increasing the 110-week survival rate of DR rats.

Effect of the AIN-93M purified diet and dietary restriction on survival in Sprague-Dawley rats: implications for chronic studies.

Survival, growth and dietary intake (DI) variables were monitored in a chronic 114-wk study in which male Sprague-Dawley rats [n = 120; National Center for Toxicological Research (NCTR) colony] consumed the AIN-93M purified diet ad libitum (AL), or an amount reduced by 31% of total AL intake inclusive of all macro- and micronutrients. The main objectives were to ascertain the survival characteristics of rats fed the AIN-93M diet and to determine whether dietary restriction (DR) increases longevity of rats fed this casein-based diet compared with the use of mixed-protein sources of the NIH-31 cereal-based diet in an earlier study. Body, liver, brain, the brain/body ratio, spleen, thymus and kidney weights, body length and body density were decreased (P < 0.05) by DR, whereas testis weight and skull length were not altered by DR. Significant age effects at 58 and 114 wk were found for body, brain, the brain/body ratio, liver and testis weights, and body density. Survival rates for the AL and 31% DR groups were 43.3 and 57.5%, respectively. Survival curves were not significantly different. The survival rate for AL rats fed the AIN-93M diet was not different from that of AL rats fed the NIH-31 diet (43.3 and 51.7%, respectively). However, the survival rate for 31% DR rats fed the AIN-93M diet was significantly lower than 25% DR rats fed the NIH-31 diet (57.5 and 87.5%, respectively) although both groups had similar body weights and energy intake at various ages. Nutritional components in the NIH-31 diet that are missing and/or reduced in the AIN-93M diet may interact with DR to increase 114-wk survival. Although the survivability, growth and anatomical results of this study suggest that the AIN-93M diet is suitable for chronic rodent studies, additional studies such as comprehensive histopathologic and physiologic investigations must be undertaken to complete the evaluation process.

Alteration in Electroencephalogram and Monoamine Concentrations in Rat Brain following Ibogaine Treatment.

Ibogaine (IBO) is a psychoactive indole alkaloid that has antiaddictive properties. However, treatment with IBO may lead to neurotoxicity, since IBO and its metabolites interact persistently with many neurotransmitter systems. Here, we recorded cortical electroencephalogram (EEG) signals from rats anesthetized with isoflurane. The heart rate (HR) was monitored via electrocardiogram (EKG) electrodes. After the baseline EEG was recorded, rats received one intraperitoneal (i.p.) dose of 50 mg/kg IBO. EEG signals were recorded for 2 hr. Rats were then sacrificed and brains dissected into frontal cortex (FC), caudate nucleus (CN), hippocampus (HIP), and brain stem (BS). The level of dopamine (DA), serotonin (5-HT), and their metabolites were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Compared with baseline, a decrease in HR immediately after IBO injection and a decrease in δ, θ, α, and ß power spectra frequency bands (1-4, 4-8, 8-13, 13-32Hz) during the first 30 min after IBO administration was observed. EEG recovered within the next 15 min. In CN, the level of DA decreased and DA turnover rate increased significantly. The levels of 5-HT increased in FC. The pattern of EKG and EEG response to IBO may be due to multiple receptor interactions of IBO.