Two Hits of EDCs Three Generations Apart: Effects on Social Behaviors in Rats, and Analysis by Machine Learning

All individuals are directly exposed to extant environmental endocrine-disrupting chemicals (EDCs), and indirectly exposed through transgenerational inheritance from our ancestors. Although direct and ancestral exposures can each lead to deficits in behaviors, their interactions are not known. Here we focused on social behaviors based on evidence of their vulnerability to direct or ancestral exposures, together with their importance in reproduction and survival of a species. Using a novel “two hits, three generations apart” experimental rat model, we investigated interactions of two classes of EDCs across six generations. PCBs (a weakly estrogenic mixture Aroclor 1221, 1 mg/kg), Vinclozolin (antiandrogenic, 1 mg/kg) or vehicle (6% DMSO in sesame oil) were administered to pregnant rat dams (F0) to directly expose the F1 generation, with subsequent breeding through paternal or maternal lines. A second EDC hit was given to F3 dams, thereby exposing the F4 generation, with breeding through the F6 generation. Approximately 1200 male and female rats from F1, F3, F4 and F6 generations were run through tests of sociability and social novelty as indices of social preference. We leveraged machine learning using DeepLabCut to analyze nuanced social behaviors such as nose touching with accuracy similar to a human scorer. Surprisingly, social behaviors were affected in ancestrally exposed but not directly exposed individuals, particularly females from a paternally exposed breeding lineage. Effects varied by EDC: Vinclozolin affected aspects of behavior in the F3 generation while PCBs affected both the F3 and F6 generations. Taken together, our data suggest that specific aspects of behavior are particularly vulnerable to heritable ancestral exposure of EDC contamination, that there are sex differences, and that lineage is a key factor in transgenerational outcomes.

Sex differences in rat renal arterial responses following exercise training

During aerobic exercise, hemodynamic alterations occure; while blood flow in skeletal muscle arteries increases, it decreases in visceral vessels due to mesenterial vasoconstriction. However, maintaining renal blood flow during intensive sport is also a priority. Our aim was to investigate the changes of vascular reactivity and histology of isolated renal artery of male and female rats in response to swim-training. Wistar rats were distributed into four groups: male sedentary (MSed), male trained (MTr), female sedentary (FSed), and female trained (FTr). Trained animals underwent a 12-week-long intensive swimming program. Vascular function of isolated renal artery segments was examined by wire myography. Phenylephrine-induced contraction was lower in FSed compared to MSed animals, and it was decreased by training in male but not in female animals. Inhibition of cyclooxygenases by indomethacin reduced contraction in both sedentary groups, and in MTr but not in FTr animals. Inhibition of nitric oxide production increased contraction in both trained groups. Acetylcholine induced relaxation was similar in all experimental groups showing predominant NO-dependency. Elastin and smooth muscle cell actin density was reduced in female rats after aerobic training. This study shows that, as a result of 12-weeks-long training, there are sex differences in renal arterial responses following exercise training. Swimming moderates renal artery vasoconstriction in male animals, while it depresses elastic fiber and smooth muscle actin density in females.

Hypoxia-Ischemia During a Neonatal Sensitive Period Alters Purkinje Neuron Dendritic Complexity Differently in Male and Female Rats

Perinatal hypoxia-ischemia (HI) is a major health issue with no effective therapies beyond head cooling. Notably, male infants are at a greater risk for HI and exhibit more extreme deficits than females. Extensive clinical evidence indicates that perinatal HI impacts the developing cerebellum, yet this region has been largely ignored in preclinical models. Using a modified version of the Rice-Vannucci rat model for HI injury at postnatal day 10, we find reductions in dendritic complexity of Purkinje neurons in males one week later. Females exhibited modest but opposite effects, with slight increases in dendritic complexity, based on Sholl analysis. A custom-made NanoString panel for quantifying mRNAs associated with development, inflammation, and sex differences found almost no commonality in the response to HI in males versus females, with males up-regulating genes associated with microglia activity whereas females increased expression of a protective complement protein, but also of enzymes associated with endocannabinoids and prostaglandins. Both sexes exhibited a reduction in the GABA-synthetic enzymes, GAD-65 and GAD-67, after HI, suggesting increased excitotoxicity, but why males suffered more damage to the Purkinje neurons is unknown.

Preclinical toxicity test results of a new antiviral–immune-modulator compound consisting of flavonoid molecules (COVID-19 clinical trial preliminary data)

Aim: Isolated specific glycone–aglycone conjugated flavonoids which are investigated for their effect of bioavailability and molecular concentrations. The specific formula is then tested via in vitro and in vivo cytotoxicity tests. Methods: Considering the higher affinity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), quercetin, quercetin 3-sambubioside-3’-glucoside, luteolin, apigenin-7-4’alloside, kaempferol-7-O-glucoside, epicatechin-epigallocatechin-3-O-gallate, and hesperetin were selected to investigate the effects of a new combination of the formula. Specific chemical analyses, such as high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS), quadrupole time of flight mass spectrometry (QTOF–MS) analysis and ultraviolet–visible (UV–VIS) spectrophotometry, were performed for molecular qualification and quantification. Results: In silico molecular docking analyses have shown that flavonoids can bind strongly to the spike protein and main protease of the SARS-CoV-2 virus. Flavonoids also have anti-inflammatory and immune-modulating activity by inhibiting cytokines. Although flavonoids may be a treatment alternative for coronavirus disease 2019 (COVID-19), an effective flavonoid compound has yet to be developed. The main problem here is that the absorption rate of flavonoids is very low (2–10%) in the intestines, and these compounds are metabolized rapidly. In contrast, according to recent literature, a conjugated flavonoid mixture is better absorbed in the small intestine, and its toxic effects are relatively fewer. Conclusions: It is found that the new formula has no cytotoxic or genotoxic effects. Furthermore, oral administrations of the new compound did not produce any toxicity symptoms or any mortality in male and female rats. The pre-clinical in vitro and in vivo toxicity test results indicated that the new flavonoid formula can be safely used for clinical trials.

CHARACTERISTICS OF REDOX STATUS IN MALE AND FEMALE RATS WITH EXPERIMENTAL DIABETES MELLITUS, GUERIN'S CARCINOMA AND ASSOCIATED PATHOLOGY

Taking into account cancer and diabetes comorbidity, the role of free radicals in these conditions and the dependence of pathological processes on the gender of animals, the aim of the study was to examine lipid peroxidation (LPO) intensity and the activity of the key antioxidant enzyme superoxide dismutase (SOD) in the heart, kidneys, liver, and in tumors of male and female rats with diabetes mellitus (DM), Guerin's carcinoma (GC) or both diseases. Materials and Methods. The study included 80 white nonlinear male and female rats (180–220 g). The animals were divided into 4 groups, each group included 10 animals (either male or female). At the time of GC inoculation, the blood glucose level in animals with alloxan diabetes was 25.4±1.2 mmol/L. The authors used conventional spectrophotometric methods to examine the content of malondialdehyde (MDA), diene conjugates (DC), and SOD activity. Statistical analysis was performed using Statistical analysis software Statistica 10.0. Results. The most pronounced changes in the studied parameters were found in the heart of female rats with isolated GC and associated diabetes mellitus: more than a threefold increase in MDA, a significant increase in DC against the background of an increase in SOD activity (by 5.5–6.3 times in comparison to intact animals). The amount of MDA in GC tissue depended on the tumor size: the maximum increase in both parameters was observed in male rats with GC growth and associated DM. Conclusion. The changes in the content of LPO products and SOD activity in the heart, kidneys, and liver of rats with diabetes mellitus and tumor growth depend on the type of the examined tissue and the gender of the animals. Disorders of the redox status found in the heart tissue can make a significant contribution to cardiopathology, which is often observed in diabetes mellitus and malignant tumor growth. Key words: diabetes mellitus, Guerin's carcinoma, rats, combined pathology, heart, kidneys, liver, malondialdehyde, diene conjugates, superoxide dismutase. Учитывая коморбидность рака и диабета, роль свободнорадикальных процессов при этих состояниях и зависимость течения патологических процессов от пола животных, целью исследования явилось изучение интенсивности перекисного окисления липидов (ПОЛ) и активности ключевого антиоксидантного фермента супероксиддисмутазы (СОД) в сердце, почках, печени, а также в опухоли крыс разного пола при сахарном диабете (СД), карциноме Герена (КГ) и при их сочетании. Материалы и методы. В исследование были включены 80 белых нелинейных крыс обоего пола массой 180–220 г, разделенных на 4 группы по 10 животных каждого пола. На момент перевивки КГ уровень глюкозы в крови животных с аллоксановым СД составил 25,4±1,2 ммоль/л. Содержание малонового диальдегида (МДА), диеновых конъюгатов (ДК) и активность СОД исследованы общепринятыми спектрофотометрическими методами. Статистический анализ проведен с использованием программы Statistica 10.0. Результаты. Наиболее выраженные изменения изученных показателей выявлены в сердце самок при изолированной КГ и КГ, растущей на фоне СД: более чем трехкратное увеличение МДА, значимый прирост ДК на фоне повышения активности СОД в 5,5–6,3 раза относительно интактных животных. В ткани КГ прослеживалась зависимость степени выраженности увеличения содержания МДА от размеров опухоли: максимальное увеличение обоих показателей наблюдалось у самцов при росте КГ на фоне СД. Выводы. Направленность изменения содержания продуктов ПОЛ и активности СОД в сердце, почках и печени крыс при СД и опухолевом росте зависит от типа исследованной ткани и пола животных. Обнаруженные в ткани сердца нарушения редокс-статуса могут вносить значимый вклад в развитие кардиопатологии, часто наблюдаемой при СД и злокачественном росте. Ключевые слова: сахарный диабет, карцинома Герена, крысы, сочетанная патология, сердце, почки, печень, малоновый диальдегид, диеновые конъюгаты, супероксиддисмутаза.

Evaluation of Some Male and Female Rats’ Reproductive Hormones Following Administration of Aspartame With or Without Vitamin C or E

Aspartame (ASP) is a sugar substitute. Its use rose because it has been demonstrated to have deleterious effects after being metabolized. In the presence of antioxidant vitamins C or E, the effects of ASP on reproductive hormones of adult male and female Albino Wister rats were investigated. A total of eighty male and female rats were used in this study. The rats were divided into four groups: group 1, received no treatment; group 2, received ASP at 40 mg/kg BW; group 3, received ASP at 40 mg/kg BW with vitamin C at 150 mg/kg BW; and group 4, received ASP at 40 mg/kg BW and vitamin E at 100 mg/kg BW. All treatments were given orally by gavage needle once daily for consecutive 90 days. The levels of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone hormone (TH) were measured after 90 days in blood plasma. In comparison with the control group, ASP treatment resulted in lower levels of E2, FSH, and LH in male and female rats. When the antioxidants vitamin C or E was given, the effects of ASP were reversed, and the levels of E2, LH, and FSH were increased. The testosterone hormone was likewise significantly increased by ASP, but testosterone hormone concentrations were decreased by vitamin C or E treatments. Long-term ASP consumption caused interfering with testicular and ovarian hormonal activity, while vitamins C and E on the other hand, overcome longstanding consumption ASP's effects.

Δ9-tetrahydrocannabinol (THC) vapor exposure produces conditioned rewarding effects in male and female rats

Background: Vaping of cannabis and cannabis extracts containing Δ9-tetrahydrocannabinol (THC, the primary psychoactive constituent of cannabis) is on the rise. Development of animal models using vapor exposure is important for increasing our understanding of the rewarding and aversive effects of vaped cannabinoids. Currently there are limited data on the conditioned rewarding effects of THC vapor in rats, and no studies to date examining sex differences. Methods: Male and female Sprague-Dawley rats (N=96; 12 per sex/group) underwent place conditioning after exposure to vaporized THC or vehicle (propylene glycol, PG). THC vapor-conditioned rats received one of three THC vapor exposure amounts (THC Group 1: 5 puffs of 100 mg/ml THC, THC Group 2: 5 puffs of 200 mg/ml THC, or THC Group 3: 10 puffs of 200 mg/ml THC) and matched vehicle vapor (PG) exposure and on alternate days for two sets of 8 daily sessions (16 days total). Vehicle-conditioned rats (Veh Group 0) received only PG vapor exposure each day. Rats were passively administered vapor for 30-min immediately before daily, 30-min conditioning sessions. Untreated rats completed place preference tests after the 8th and 16th conditioning sessions and then testing continued daily until extinction occurred. Following extinction, rats underwent a THC vapor-primed reinstatement session. Results: Male and female rats showed an exposure-dependent preference for the THC vapor-paired chamber, though sex differences were observed. The lowest THC vapor exposure group tested (THC Group 1) did not produce CPP in males or females. Exposure to the middle condition tested (THC Group 2) resulted in preference for the THC vapor-paired chamber in males, but not females. The highest THC vapor exposure condition tested (THC Group 3) produced place preference in both males and females. Preference for the THC-paired chamber extinguished more quickly in males than in females. Following extinction, THC vapor re-exposure (i.e., drug-prime) did not result in reinstatement of place preference for either sex. Conclusion: This study demonstrated conditioned rewarding effects of THC vapor in both male and female rats, and provides evidence for sex differences in doses of THC vapor that produce CPP and in time to extinction. Conditioned place aversion was not observed at any of the THC vapor exposure amounts tested. These data provide a foundation for future exploration of the conditioned rewarding effects vaporized THC, cannabis and its constituents in preclinical models.

Low Dietary n6/n3 Ratio Attenuates Changes in the NRF 2 Gene Expression, Lipid Peroxidation, and Inflammatory Markers Induced by Fructose Overconsumption in the Rat Abdominal Adipose Tissue

The objective of this study was to examine the benefits of different n6/n3 polyunsaturated fatty acid ratios on the lipid metabolism, insulin resistance, and oxidative stress in the adipose tissue of rats fed a high-fructose diet. Male and female rats were divided into four groups: a control group (CON) (n6/n3 ratio ~7), a high-fructose group (HF) (n6/n3 ratio ~7), an N6-HF group (n6/n3 ratio ~50), and the DHA-HF group (n6/n3 ratio ~1, with the addition of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid). The CON group received plain water and the HF group received 15% fructose in their drinking water. Fructose induced an increase in the content of serum triglycerides, serum cholesterol, and HOMA-IR index. Among the fatty acids, elevated proportions of C18:1n9 and C16:1n7, as well as an increase in total monounsaturated fatty acid (MUFA), were found in the adipose tissue of the HF group. Fructose treatment also changed oxidative parameters, including a marked increase in the serum malondialdehyde (MDA) content. Meanwhile, DHA supplementation caused a significant decrease in the serum MDA concentration in comparison with the HF group. In addition, DHA/EPA supplementation attenuated oxidative stress by increasing NRF 2 gene expression. Fructose treatment also significantly decreased the adiponectin level, while DHA supplementation ameliorated it. The changes observed in this trial, including the decrease in the content of DHA and EPA, the decreased EPA/ARA ratio, and the increase in the expression of inflammatory genes, are characteristics of the low-grade inflammation caused by fructose treatment. These changes in the rat adipose tissue could be prevented by dietary intervention consisting of DHA supplementation and a low n6/n3 ratio.