Female F344 Rats Research Articles

Dantrolene Attenuates Cardiotoxicity of Doxorubicin Without Reducing its Antitumor Efficacy in a Breast Cancer Model

Dysregulation of calcium homeostasis is a major mechanism of doxorubicin (DOX)-induced cardiotoxicity. Treatment with DOX causes activation of sarcoplasmic reticulum (SR) ryanodine receptor (RYR) and rapid release of Ca2+ in the cytoplasm resulting in depression of myocardial function. The aim of this study was to examine the effect of dantrolene (DNT) a RYR blocker on both the cardiotoxicity and antitumor activity of DOX in a rat model of breast cancer. Female F344 rats with implanted MAT B III breast cancer cells were randomized to receive intraperitoneal DOX twice per week (12 mg/kg total dose), 5 mg/kg/day oral DNT or a combination of DOX + DNT for 3 weeks. Echocardiography and blood troponin I levels were used to measure myocardial injury. Hearts and tumors were evaluated for histopathological alterations. Blood glutathione was assessed as a measure of oxidative stress. The results showed that DNT improved DOX-induced alterations in the echocardiographic parameters by 50%. Histopathologic analysis of hearts showed reduced DOX induced cardiotoxicity in the group treated with DOX + DNT as shown by reduced interstitial edema, cytoplasmic vacuolization, and myofibrillar disruption, compared with DOX-only–treated hearts. Rats treated with DNT lost less body weight, had higher blood GSH levels and lower troponin I levels than DOX-treated rats. These data indicate that DNT is able to provide protection against DOX cardiotoxicity without reducing its antitumor activity. Further studies are needed to determine the optimal dosing of DNT and DOX in a tumor-bearing host.

Cypermethrin exposure during perinatal period affects fetal development and impairs reproductive functions of F1 female rats

Cypermethrin (CYP) is a ubiquitously present synthetic pyrethroid insecticide. It has endocrine disrupting activities which may adversely affect reproductive development and functions of offspring if exposed during critical developmental period. The present study was undertaken to delineate the effects of CYP exposure in pregnant female rats during perinatal period on the sexual maturation, hormonal regulation, reproductive development and fertility of F1 female offspring and its molecular mechanism of action. Pregnant rats (F0) were gavaged daily with 0, 1, 10, 25 mg/kg bw/day CYP and 10 μg/kg bw/day Diethylstilbestrol (DES; positive control) from gestation day (GD) 6 to postnatal day (PND) 21. The reproductive development and function parameters were evaluated at PND 45 and 75. Reduced body weight, delayed vaginal opening, and disrupted estrous cyclicity were observed at 25 mg/kg CYP dose. CYP exposure significantly affected the reproductive organ development and their functions at all doses. Significant alterations in ovarian and uterine histology such as luteinization, reduction of primordial follicular reserves, presence of multi-oocyte follicles and thin degenerative luminal and glandular uterine epithelium were observed at adulthood. Altered circulatory steroid hormone levels and expression of ovarian and uterine steroid hormone receptors were observed at PND 75 in the F1 female offspring. Expression of HOXA10 and α-SMA which are important for uterine integrity and functions, were found to be altered at PND 75. Increased pre-implantation loss (PIL%), post-implantation loss (POL%), and reduced litter size in F1 females when cohabitated with unexposed fertile male rats were observed. Overall, perinatal exposure of pregnant rats to CYP led to significant long lasting effects on the reproductive functions of F1 female offspring. The adverse effects were passed on to F2 generation via female germ line and posed developmental anomalies. The present finding necessitates additional molecular studies to understand its trans-generational mechanism of action via female germline.

Thirteen-week inhalation toxicity study of 1-methylnaphthalene in F344 rats.

1-Methylnaphthalene is generally utilized in solvents, as an intermediate in organic synthesis, a dye carrier, in resins, and others. There are some toxicological studies of 1-methylnaphthalene; however, inhalation toxicity studies are rare. Each of 10 male and female F344 rats was exposed to vapors of 1-methylnaphthalene for 13weeks (6h a day, 5days per week) at concentrations of 0, 0.5, 4, and 30ppm in a whole-body inhalation chamber system. The exposure concentrations were 0.52 ± 0.05, 4.08 ± 0.25, and 30.83 ± 1.28ppm for the low-, middle-, and high-dose group, respectively. Body weight changes were not affected by exposure to 1-methylnaphthalene. Blood prothrombin time was delayed at 30ppm in male and female groups, and activated partial thromboplastin time was also delayed at 30ppm in the male group. Values of alanine aminotransferase in the serum were decreased and those of albumin were increased at 30ppm in the male group. Differential cell counts and levels of lactate dehydrogenase in the bronchoalveolar lavage fluid were not affected. However, mucous cell hyperplasia in the nasopharyngeal tissues was found and the severity was correlated to exposure concentrations. In conclusion, 1-methylnaphthalene mainly affects the upper respiratory system and the no-observed-adverse-effect levelis suggested to be 4ppm on the basis of histopathological findings.

Paternal morphine exposure induces bidirectional effects on cocaine versus opioid self-administration

The United States is in the midst of an opioid epidemic and is thus experiencing unprecedented levels of opioid exposure. A growing body of evidence has demonstrated that this may have consequences on multiple generations. The current set of experiments examined the effect of male adolescent opioid exposure on cocaine and opioid self-administration in the F1 generation. Male Sprague Dawley rats were administered increasing doses of morphine (5–25 mg/kg, s.c.) for 10 days during adolescence (P30-39). Rats were then maintained drug free until adulthood (P70-80) at which point they were mated with drug-naïve females. Male and female F1 offspring were first examined for cocaine self-administration during adulthood. Naïve littermates were tested for morphine self-administration acquisition followed by a within subjects design progressive ratio test for morphine, oxycodone, and cocaine. Results show that male and female F1 rats have delayed acquisition and decreased intake of cocaine. In addition, they have blunted PR levels compared to Sal-F1 control rats. Female Mor-F1 rats also demonstrate increased levels of morphine intake during acquisition and increased PR responding for oxycodone. Surprisingly, even following acquisition of morphine self-administration, Mor-F1 males and females still demonstrate blunted effort for cocaine. There were no differences in sucrose self-administration in naïve littermates. MorF0 seminiferous tubules demonstrated increased levels of acetylated histone H3 and there were increased levels of BDNF mRNA in the mPFC in male and female F1 offspring. Together, these data identify systems that are vulnerable to the impact of opioids in the F0 generation.

The influence of long-term drinking of citric acid on the organism of F0 female rats

The influence of long-term drinking of Citric acid on the organism of female rats was studied by the investigation of morphological and biochemical indices and organ mass coefficients. The study was performed on 10 laboratory female rats F0, which were organized in groups (5 – control, 5 – experimental) 2–2.5 months aged, with body mass 110–120 g. The animals of the experimental group received aqueous solution of Citric acid (С6Н12О6 х Н2О, “ch.p.”) in the dose 0.8 mg/kg b. m. during the physiologic and puberty periods, fertilization and pregnancy. Animals of both groups had constant access to the drinking water. The received results of studies of red cells in the experimental group are comparative to the control group animals indicate the tendency to decrease the number of red blood cells by 6%, hemoglobin in the blood on 22.7%, average hemoglobin concentration in hemoglobin by 25%, average hemoglobin content in the erythrocyte on 20.9%, average hemoglobin volume on 4% against the background of an increase of hematocrit value on 5.4%. The experimental data obtained indicate a probable increase in the number of leukocytes in the blood of the experimental group of animals on 42.4%, and a tendency for a 2-folf increase in eosinophils. The results of biochemical studies of serum indicate minor differences between serum iron content (increase on 5.6%) in experimental and control animals groups. Also, the received results indicate the tendency of an increase by 3.3% of serum trasferrin on the background of decrease of total (by 3.6%) and unsaturated (by 7.6%) Fe-binding ability of blood. Animals receiving Citric acid found a tendency to increase, in terms of indicators of ALT activity – by 18.9% and alkaline phosphatase by 13.2% comparing with the control group of animals. The blood of female rats showed significantly higher urea level of 72.5% and a lower creatinine level of 19.6%. The content of average molecular weight substances in the serum of female rats of the experimental group under the influence of Citric acid is less than in animals of the control group on 2.8%. Studies of the individual organs status with the determination of their weight and mass coefficients revealed that against a background to the coefficients of the control group of female rats the weight of the liver increased likely by 16.1%. In the future, we plan to investigate the effect of long-term drinking of Citric acid on the organism of F1 rats.

Promotion effects of acetoaceto-o-toluidide on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder carcinogenesis in rats.

Recent epidemiological studies have indicated that occupational exposure to the aromatic amine acetoaceto-o-toluidide (AAOT) was associated with a marked increase in urinary bladder cancers in Japan. However, little is known about the carcinogenicity of AAOT. To evaluate the urinary bladder carcinogenicity of AAOT, male and female F344 rats were treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4weeks followed by dietary administration of 0, 0.167, 0.5, or 1.5% AAOT for 31weeks. The incidences and multiplicities of bladder tumors were significantly increased in the 0.5 and 1.5% groups of male and female rats in a dose-response manner. AAOT and seven downstream metabolites were detected in the urine of the male and female rats administered AAOT with levels increasing in a dose-dependent manner. The most abundant urinary metabolite of AAOT was the human bladder carcinogen o-toluidine (OTD), which was at least one order of magnitude higher than AAOT and the other AAOT metabolites. In a second experiment, male F344 rats were administered 0, 0.167, or 1.5% AAOT for 4weeks. Gene expression analyses revealed that the expression of JUN and its downstream target genes was increased in the urothelium of male rats treated with 1.5% AAOT. These results demonstrate that AAOT promotes BBN-induced urinary bladder carcinogenesis in rats and suggest that overexpressed of JUN and its downstream target genes may be involved the bladder carcinogenicity of AAOT. In conclusion, AAOT, like other carcinogenic aromatic amines, is likely to be a carcinogen to the urinary bladder, and OTD metabolized from AAOT is the ultimate carcinogen.

Cadmium exposure during prenatal development causes testosterone disruption in multigeneration via SF-1 signaling in rats

The mechanisms of prenatal cadmium (Cd) exposure cause adverse effect transmission to future generations that remain unclear. In this study, pregnant SD rats were orally dosed with Cd (0, 0.5, 2.0, and 8.0 mg/kg/day) from gestation day 1 until birth. F1 female rats were mated with untreated males for F2 generation. In both generations, after prenatal Cd exposure, histopathological examinations showed testicular development disorder. A significant decrease in serum testosterone (T) was observed in the F1 rats, but a significant increase in serum T was observed in the F2 rats. Moreover, both the F1 and F2 rats had different patterns of mRNA and protein expression for testicular steroidogenic factor 1 (SF-1) and steroidogenic enzymes at postnatal days (PNDs) 21 and 56. We also found that rno-miR-328a-5 and rno-miR-10b-5p significantly changed and TargetScan software showed that both of these microRNAs targeted SF-1 and steroidogenic acute regulatory (StAR), respectively. Overall, the results indicate that prenatal Cd exposure causes male reproductive problems in a multigenerational manner. In addition, SF-1 signaling was disrupted and the expressions of microRNAs were affected, which may be an important target for Cd-induced reproductive toxicity in offspring.

Prenatal Exposure to Environmentally-Relevant Contaminants Perturbs Male Reproductive Parameters Across Multiple Generations that are Partially Protected by Folic Acid Supplementation

The paternal environment is thought to influence sperm quality and future progeny may also be impacted. We hypothesized that prenatal exposure to environmentally-relevant contaminants impairs male reproduction, altering embryo gene expression over multiple generations. Folic acid (FA) can improve sperm quality and pregnancy outcomes, thus we further hypothesized that FA mitigates the contaminants. Sprague-Dawley F0 female rats treated with persistent organic pollutants (POPs) or corn oil and fed basal or supplemented FA diets, then used to yield four generations of litters. Only F0 females received POPs and/or FA treatments. In utero POPs exposure altered sperm parameters in F1, which were partly rescued by FA supplementation. Paternal exposure to POPs reduced sperm quality in F2 males, and the fertility of F3 males was modified by both POPs and FA. Ancestral FA supplementation improved sperm parameters of F4 males, while the POPs effect diminished. Intriguingly, F3 males had the poorest pregnancy outcomes and generated the embryos with the most significantly differentially expressed genes. Early-life exposure to POPs harms male reproduction across multiple generations. FA supplementation partly mitigated the impact of POPs. The two-cell embryo transcriptome is susceptible to paternal environment and could be the foundation for later pregnancy outcomes.

Pathological Study on Epithelial-Mesenchymal Transition in Silicotic Lung Lesions in Rat

Silicosis, caused by the inhalation of crystalline silicon dioxide or silica, is one of the most severe occupational diseases. Persistent inflammation and progressive massive pulmonary fibrosis are the most common histological changes caused by silicosis. Association of epithelial-mesenchymal transition (EMT) of hyperplastic type II epithelial cells with the fibrotic events of pulmonary fibrosis has been suggested in in vitro silica-exposed cultured cell models, patients with idiopathic pulmonary fibrosis, and bleomycin-induced experimental models. Histological features of EMT, however, are not fully described in silicotic lungs in in vivo. The purpose of this study was to demonstrate EMT of hyperplastic type II epithelial cells in the developmental process of progressive massive pulmonary fibrosis in the lungs of rats exposed to silica. F344 female rats were intratracheally instilled with 20 mg of crystalline silica (Min-U-Sil-5), followed by sacrifice at 1, 3, 6, and 12 months after instillation. Fibrosis, characterized by the formation of silicotic nodules, progressive massive fibrosis, and diffuse interstitial fibrosis, was observed in the lungs of the treated rats; the effects of fibrosis intensified in a time-dependent manner. Hyperplasia of the type II epithelial cells, observed in the massive fibrotic lesions, dominated in the lungs of rats at 6 and 12 months after the treatment. Immunohistochemistry of the serial sections of the lung tissues demonstrated positive labeling for cytokeratin, vimentin, and α-smooth muscle actin in spindle cells close to the foci of hyperplasia of type II epithelial cells. Spindle cells, which exhibited features of both epithelial cells and fibroblasts, were also demonstrated with bundles of collagen fibers in the fibrotic lesions, using electron microscopy. Increased expression of TGF-β was shown by Western blotting and immunohistochemistry in the lungs of the treated rats. These findings suggested that enhanced TGF-β expression and EMT of hyperplastic type II epithelial cells are involved in the development process of progressive massive pulmonary fibrosis during silicosis.

Sexually Dimorphic Effects of Dietary Vitamin D3 Supplementation on Cognition and the Gut Microbiome in Aging Rats (P14-006-19)

ObjectivesIncreasing evidence suggests that vitamin D plays a role in maintaining cognitive function and that vitamin D deficiency may accelerate age-related cognitive decline. Here, we determined if a long-term enhanced vitamin D (VitD3, cholecalciferol) diet, higher than the standard dietary level, maintains or improves cognitive function in aging male and female rats. We also examined if the high VitD3 diet affected the gut microbiome. MethodsBeginning at 12 months of age 20 male and 20 female F344 rats were fed an AIN-93 diet containing either standard (1000 IU/kg diet) or higher (10,000 IU/kg) VitD3 for 6 months. The Morris water maze (MWM) was then used to assess learning and memory. Following the MWM, the gut microbiome from undigested chyme collected from the intestinal cecum was identified and taxonomically classified by Argonne National Laboratory using 16S rRNA sequences. ZRT Laboratory determined 25-(OH)VitD3 levels from cardiac blood. A two-way ANOVA and Tukey post-hoc was used to test for statistical significance. ResultsAfter 3 days of training the probe test on day 4 indicated that the higher VitD3 diet significantly reduced path length and latency (P = 0.01) to the digital platform in females but not males. On day 5 platform location was changed and animals received one day of reversal training. On day 8, three days after reversal training, the reversal probe indicated that higher dietary VitD3 improved performance in males but not females by significantly reducing path length and latency to the digital platform (P < 0.05). Analyses of the cecal microbiome content indicated that for numerous bacteria sex specific differences were present. Further, the Shannon Diversity Index of the gut microbiome indicated a significant treatment effect of higher dietary VitD3 in females (P = 0.01). The higher VitD3 diet significantly elevated 25-(OH)VitD3 blood levels. ConclusionsThese results indicate that a high VitD3 diet may preserve cognitive acuity during aging. Further, VitD3 may have sexually dimorphic effects on memory formation. The present results replicate our previous study that a high VitD3 diet preserves cognition in aging male rats (Latimer et al. 2014). The microbiome showed sexually dimorphic differences and the high VitD3 diet appeared to affect the microbiome in females more than males. The significance of this is not clear. Funding SourcesNIA, NIDDK.

Aging effects on oxidative capacity in type‐identified diaphragm muscle fibers

The diaphragm muscle (DIAm) comprises four muscle fiber/ motor unit types: Fatigue resistant type S and FR motor units with type I and IIa fibers, respectively that are recruited for ventilatory behaviors; and more fatigable type FInt and FF motor units with type IIx and/or IIb fibers that are only recruited during high force expulsive/straining behaviors of the DIAm. With advanced aging in Fischer 344 rats (F344), type IIx and/or type IIb DIAm fibers are vulnerable to sarcopenia (reductions in specific force and cross‐sectional area, CSA), while type I and IIa fibers are resilient. This is consistent with the fact that ventilatory behaviors are conserved with aging, while airway clearance behaviors are compromised. Throughout life, breathing imposes a high duty cycle (&gt;33 %) of DIAm activity and thus, a high energy demand for type I and IIa fibers that is met by a high oxidative capacity. However, the effect of advanced age on DIAm fiber oxidative capacity has not been examined. Accordingly, we hypothesize that the oxidative capacity in type I and IIa DIAm fibers is not affected by age, whereas concomitant with sarcopenia, the oxidative capacity of type IIx and/or IIb fibers is reduced.To assess DIAm fiber oxidative capacity, we measured succinate dehydrogenase (SDH) activity in type‐identified DIAm fibers from 6, 18 and 24‐month old male and female F344 rats. Following euthanasia, the DIAm was excised and 2–3 mm strips from the mid‐costal region were dissected, stretched to optimal length and rapidly frozen in melting isopentane. Serial transverse sections (6 μm) were cut to assess fiber type (based on myosin heavy chain, MyHC isoform), cross‐sectional area (CSA) and SDH activity. For SDH, sections were incubated at 25°C for 8 min in solutions containing 0 or 80 mM succinate and 1.5 mM nitro blue tetrazolium (reaction indicator), 5 mM EDTA, 0.2 mM PMS and 0.1 mM azide in 0.1 M phosphate buffer. Images were acquired every 5 s across a 15‐min period using a 20x objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction (change in optical density‐ OD) was confirmed across the 15‐min period for each fiber type.The presence of DIAm sarcopenia was confirmed in 24‐month old rats by a 25 % decrease in specific force and a 20% decrease in type IIx and/or IIb CSA. At all ages, the SDH activity of type I and IIa fibers was higher than type IIx/IIb fibers. No age‐related difference was observed in the SDH activity of type I or IIa fibers, whereas SDH activity of type IIx and/or IIb DIAm fibers was reduced at 24 months compared to younger animals.These results underscore the remarkable vulnerability of IIx and/or IIb fibers to sarcopenia, with profound resilience in I and IIa fibers. We propose that mitochondrial abnormalities may underlie the selective vulnerability of IIx and/or IIb fibers possibly due to changes at the phrenic motor neuron level.Support or Funding InformationSupported by NIH grant AG44615This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acetoaceto-o-Toluidide Enhances Cellular Proliferative Activity in the Urinary Bladder of Rats

Acetoaceto-o-toluidide (AAOT) is made from ortho-toluidine (OTD) and is used for the synthesis of pigments. A report of occupational urinary bladder carcinomas in Japanese workers chronically exposed to OTD and AAOT has recently been published. OTD is a well-known human urinary bladder carcinogen; however, little is known about the toxicity and the carcinogenicity of AAOT. The aim of the present study is to evaluate the toxic effects of AAOT on urinary bladder epithelium. In vitro, the cytotoxicities of AAOT and OTD were evaluated in rat (MYP3) and human (1T1) urothelial cells. The LC50 of AAOT was higher than that of OTD in both MYP cells and 1T1 cells. In vivo, 6-week-old male and female F344 rats were fed diets supplemented with 0%, 1.5%, or 3% AAOT for 4 weeks. Incidences of simple hyperplasia, cell proliferative activity, and γ-H2AX expression, which is a novel marker for the prediction of carcinogenicity, were significantly increased in a dose-dependent manner in the bladder urothelium of male and female rats administered AAOT. Furthermore, in male and female rats administered AAOT, the major urine metabolite of AAOT was OTD. These results demonstrate that AAOT has proliferation-enhancing activity and suggest that OTD metabolized from AAOT may play a pivotal role in the deleterious effects of AAOT in rats. The results of the present study also indicate that AAOT, like other carcinogenic aromatic amines, is likely to be a human bladder carcinogen.

Metabolism of 4-methylimidazole in Fischer 344 rats and B6C3F1 mice

4-Methylimidazole (4-MeI) is a widely used chemical, also identified as a by-product of heating foods. In cancer bioassays, 4-MeI induced lung tumors in mice, but not in rats. To establish if metabolic differences could explain species difference in carcinogenicity, this study investigated metabolism of 4-MeI in rat and mouse lung and liver microsomes and S-9 fractions, and in vivo in rats and mice. No metabolites were detected in rat or mouse lung and liver microsomes, or lung S-9 fractions. Male and female F-344 rats and B6C3F1 mice were administered 50 and 150 mg/kg [14C] 4-MeI by gavage. Excreta, exhaled CO2 and volatiles were collected for 48 h. Elimination was mainly via urine, with 79–89% of the radioactivity in urine in rats and 41–70% in mice. Most of the radioactivity (71–88%) in urine was unchanged 4-MeI. Additional radioactive peaks (the largest metabolite was 8–18%) were characterized by LC-MS/MS as 4-hydroxymethylimidazole, its glucuronide, and other oxidized products, including methylhydantoin. 4-MeI was largely excreted unchanged in rats and mice with limited oxidative metabolism and conjugation. 4-MeI was not oxidized in subcellular fractions from rat and mouse lung and liver. Overall, the metabolism of 4-MeI appeared similar between rats and mice.

Late aging alters behavioral sensitivity to ethanol in a sex-specific manner in Fischer 344 rats

Responsiveness to ethanol (EtOH) differs as a function of age. Adolescent rodents are less sensitive than adults to the sedative effects of EtOH, whereas they show enhanced sensitivity to EtOH-induced social facilitation. Late aging is associated with a natural decline in social behavior and aging-related peculiarities in sensitivity to EtOH have been largely unexplored. Whether there are sex differences in the behavioral response to EtOH during late aging remains unknown. Thus, behavioral responses to EtOH in male and female Fischer (F) 344 rats aged 4–5 months (adult) and 19–20 months (aging) were examined. First, the effects of saline and EtOH (0.5 and 0.75 g/kg) on social interaction were assessed. Social investigation and contact behavior were lower in aging animals and higher in females. Interestingly, in aged females, social contact behavior was increased following a 0.5 g/kg EtOH dose, whereas the same dose suppressed social contact in aged males. Behavioral sensitivity to the sedative effects of 3.0 and 3.5 g/kg EtOH was assessed with the loss of righting reflex (LORR) test. Although latency to LORR did not differ as a function of age or sex, aged rats showed significantly greater LORR duration and significantly lower blood ethanol concentrations (BECs) at regaining of the righting reflex relative to adults. In addition, females had a lower LORR duration, regardless of age; no sex differences were evident in BECs at awakening. In a second experiment, blood ethanol concentrations (BECs) over time were assessed following 0.75, 1.5, and 3.0 g/kg EtOH in 3-, 12-, and 18-month-old male and female F344 rats. Aged rats had higher peak BECs following 3.0 g/kg EtOH, whereas few age or sex differences were apparent at lower doses. Taken together, these data indicate that late aging is associated with altered sensitivity to the social facilitating effects and sedative effects of EtOH.

N-butylparaben exposure during perinatal period impairs fertility of the F1 generation female rats

Parabens are a class of preservatives widely used in the majority of personal care products, cosmetics, medicines, and food products. However, current literature suggests its plausible role as an endocrine disruptor, hence the present study was undertaken to delineate the effects of n-butyl paraben on perinatally exposed F1 female rats. F0 dams were exposed subcutaneously to n-butylparaben from gestation day 6 (GD 6) to postnatal day (PND) 21 with doses of 10, 100, and 1000 mg/kg Bw/day in corn oil. The F1 female rats were monitored for pubertal development and sexual maturation through PND 30, 45, and 75; which were subsequently subjected to fertility assessment at PND 75. Perinatal exposure to n-butylparaben resulted in-a.Delayed age of vaginal opening with disturbed estrus cycle in F1 female rats.b.A significant reduction in fertility with increased pre-and post-implantation loss; impaired steroidogenesis with decreased estradiol, progesterone, and increased testosterone levels.c.An impairment in folliculogenesis with increased number of primordial follicle maturation and reduced number of corpora leutea (CL). The morphological analysis showed degenerated follicles in the ovaries and inadequate development of the myometrium in the uterus.d.Perturbed expression levels of estrogen receptors alpha (ER α), beta (ER β) and Steroidogenic acute regulatory gene (StAR) in the ovary at PND 75.This study documents impaired steroidogenesis and folliculogenesis might be the prime reason for the reduced fertility of F1 female rats. Hence, our study suggests that health monitors need to counsel potential females planning for pregnancy to avoid exposure to parabens.

Cortical AAV-CNTF Gene Therapy Combined with Intraspinal Mesenchymal Precursor Cell Transplantation Promotes Functional and Morphological Outcomes after Spinal Cord Injury in Adult Rats.

Ciliary neurotrophic factor (CNTF) promotes survival and enhances long-distance regeneration of injured axons in parts of the adult CNS. Here we tested whether CNTF gene therapy targeting corticospinal neurons (CSN) in motor-related regions of the cerebral cortex promotes plasticity and regrowth of axons projecting into the female adult F344 rat spinal cord after moderate thoracic (T10) contusion injury (SCI). Cortical neurons were transduced with a bicistronic adeno-associated viral vector (AAV1) expressing a secretory form of CNTF coupled to mCHERRY (AAV-CNTFmCherry) or with control AAV only (AAV-GFP) two weeks prior to SCI. In some animals, viable or nonviable F344 rat mesenchymal precursor cells (rMPCs) were injected into the lesion site two weeks after SCI to modulate the inhibitory environment. Treatment with AAV-CNTFmCherry, as well as with AAV-CNTFmCherry combined with rMPCs, yielded functional improvements over AAV-GFP alone, as assessed by open-field and Ladderwalk analyses. Cyst size was significantly reduced in the AAV-CNTFmCherry plus viable rMPC treatment group. Cortical injections of biotinylated dextran amine (BDA) revealed more BDA-stained axons rostral and alongside cysts in the AAV-CNTFmCherry versus AAV-GFP groups. After AAV-CNTFmCherry treatments, many sprouting mCherry-immunopositive axons were seen rostral to the SCI, and axons were also occasionally found caudal to the injury site. These data suggest that CNTF has the potential to enhance corticospinal repair by transducing parent CNS populations.

Is oxidative stress involved in the sex-dependent response to ochratoxin A renal toxicity?

Ochratoxin A (OTA) is a mycotoxin considered the most powerful renal carcinogen in rodents and classified as a possible human carcinogen. Though its mechanism of action is still unknown, indirect DNA reactivity mediated by oxidative stress has been hypothesized to play an important role. Moreover, large sex-differences have been observed in carcinogenicity studies, male rats being more sensitive than females. Male and female F344 rats were administered (p.o.) with bicarbonate or 0.5 mg OTA/kg b.w. for 7 days; or with bicarbonate, 0.21 or 0.5 mg OTA/kg b.w. for 21 days. Total glutathione (tGSH) and oxidized glutathione (GSSG) levels, glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were analysed in kidneys. The standard alkaline comet assay was used in combination with Formamidopyrimidine-DNA glycosylase (Fpg) to detect oxidized DNA bases in kidney. No biologically relevant sex-differences were observed in all the oxidative-stress related parameters analysed. Indeed, no relevant oxidative-stress related response was observed between treated animals and controls. In accordance with the similar OTA levels and histopathological changes between both sexes observed previously in the same animals, and with other oxidative-stress related parameters measured by others, results support that there are no differences between sexes in the oxidative stress response to OTA.

Hormonal changes and folliculogenesis in female offspring of rats exposed to cadmium during gestation and lactation

It has been suggested that the toxic effects of cadmium (Cd) may disrupt ovarian and uterine functions in adults. However, Cd exposure during gestation and lactation and its effects on the reproductive development in female offspring is still not clear, and the mechanisms underlying exposure toxicology remain mostly unexplored. To investigate how Cd exposure of female rats (F0) during gestation and lactation affects the reproductive development of their female offspring, we studied the steroidogenesis, folliculogenesis, puberty onset, and litter size of the first (F1) and second (F2) filial generations following F0 female rats which had been exposed to CdCl2. The mechanisms related to the early onset of puberty induced by such exposure in female offspring were explored. Maternal exposure to Cd dramatically increased the biosynthesis of steroid hormones in F1 female offspring by the activation of cAMP/PKA pathway and up-regulated expression of steroidogenesis related proteins such as StAR, CYP11A1, 3β-HSD and CYP19A1. The high levels of steroid hormones contributed to an early puberty onset, promoted the differentiation and maturation of follicles, and led to the proliferation of endometrium that resulted in a uterus weight gain. The increased number of antral follicles eventually caused a big litter size. Despite of being free from additional Cd exposure, the levels of CYP11A1 and CYP19A1 in the ovaries of F2 female rats were also high, which resulted in a high concentration of serum progesterone. These results suggested that hormonal changes induced by exposure to Cd in utero might have a lasting effect beyond the first generation. These findings may help to better understand the origin of female sexual dysfunction in the developmental stages in general.

Stereological Analysis of Microglia in Aged Male and Female Fischer 344 Rats in Socially Relevant Brain Regions

Aging is associated with a substantial decline in the expression of social behavior as well as increased neuroinflammation. Since immune activation and subsequent increased expression of cytokines can suppress social behavior in young rodents, we examined age and sex differences in microglia within brain regions critical to social behavior regulation (PVN, BNST, and MEA) as well as in the hippocampus. Adult (3-month) and aged (18-month) male and female F344 (N = 26, n = 5–8/group) rats were perfused and Iba-1 immunopositive microglia were assessed using unbiased stereology and optical density. For stereology, microglia were classified based on the following criteria: (1) thin ramified processes, (2) thick long processes, (3) stout processes, or (4) round/ameboid shape. Among the structures examined, the highest density of microglia was evident in the BNST and MEA. Aged rats of both sexes displayed increased total number of microglia number exclusively in the MEA. Sex differences also emerged, whereby aged females (but not males) displayed greater total number of microglia in the BNST relative to their young adult counterparts. When morphological features of microglia were assessed, aged rats exhibited increased soma size in the BNST, MEA, and CA3. Together, these findings provide a comprehensive characterization of microglia number and morphology under ambient conditions in CNS sites critical for the normal expression of social processes. To the extent that microglia morphology is predictive of reactivity and subsequent cytokine release, these data suggest that the expression of social behavior in late aging may be adversely influenced by heightened inflammation.

Assessing the safety of an Ephedrae Herba aqueous extract in rats: A repeat dose toxicity study

Ephedrae Herba (EH) has been used in Asian traditional herbal medicine to cure bronchial asthma, cold, flu, chills, fever, headache, nasal congestion, and cough. In this study, we evaluated the subchronic toxicity of an Ephedrae Herba aqueous extract (EHAE) in male and female F344 rats. The EHAE was administered orally daily at doses of 0, 125, 250, 500, and 1000 mg/kg bw/day for 13 weeks. Toxicological assessment was performed to determine mortality, clinical signs, and changes in body weight, food consumption, ophthalmological, urinary, hematological, and serum biochemical parameters, macroscopic and microscopic evaluations, and organ weights. We found that oral administration of EHAE to F344 rats for 13 weeks resulted in histopathological changes in the kidneys and salivary glands. In the kidneys, increased incidence and severity of tubular basophilia were observed in females administered 1000 mg/kg bw/day of the extract. In the salivary glands, acinar cell hypertrophy was observed in males administered 500 mg/kg bw/day and in both sexes administered 1000 mg/kg bw/day of the extract. All test article-treated groups of males and females administered ≥250 mg/kg bw/day showed increased absolute and relative salivary gland weights. Therefore, the NOAEL (No Observed Adverse Effect Level) was determined as 125 mg/kg bw/day for both sexes of rats under the present experimental conditions.