Male Wistar Han Rats Research Articles

Assessment of hepatic transporter function in rats using dynamic gadoxetate-enhanced MRI: a reproducibility study

ObjectivePrevious studies have revealed a substantial between-centre variability in DCE-MRI biomarkers of hepatocellular function in rats. This study aims to identify the main sources of variability by comparing data measured at different centres and field strengths, at different days in the same subjects, and over the course of several months in the same centre.Materials and methods13 substudies were conducted across three facilities on two 4.7 T and two 7 T scanners using a 3D spoiled gradient echo acquisition. All substudies included 3–6 male Wistar-Han rats each, either scanned once with vehicle (n = 76) or twice with either vehicle (n = 19) or 10 mg/kg of rifampicin (n = 13) at follow-up. Absolute values, between-centre reproducibility, within-subject repeatability, detection limits, and effect sizes were derived for hepatocellular uptake rate (Ktrans) and biliary excretion rate (kbh). Sources of variability were identified using analysis of variance and stratification by centre, field strength, and time period.ResultsData showed significant differences between substudies of 31% for Ktrans (p = 0.013) and 43% for kbh (p < 0.001). Within-subject differences were substantially smaller for kbh (8%) but less so for Ktrans (25%). Rifampicin-induced inhibition was safely above the detection limits, with an effect size of 75 ± 3% in Ktrans and 67 ± 8% in kbh. Most of the variability in individual data was accounted for by between-subject (Ktrans = 23.5%; kbh = 42.5%) and between-centre (Ktrans = 44.9%; kbh = 50.9%) variability, substantially more than the between-day variation (Ktrans = 0.1%; kbh = 5.6%). Significant differences in kbh were found between field strengths at the same centre, between centres at the same field strength, and between repeat experiments over 2 months apart in the same centre.DiscussionBetween-centre bias caused by factors such as hardware differences, subject preparations, and operator dependence is the main source of variability in DCE-MRI of liver function in rats, closely followed by biological between-subject differences. Future method development should focus on reducing these sources of error to minimise the sample sizes needed to detect more subtle levels of inhibition.

#966 Targeting cAMP in ADPKD by small molecule activation of long form PDE4 enzymes

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common hereditary kidney disorder, affecting over 12 million people worldwide. ADPKD is characterized by the uncontrolled growth of fluid-filled cysts in the kidney that can lead to kidney enlargement, loss of function, and associated complications. Currently, there are limited treatment options for ADPKD patients. By activating long form PDE4 enzymes and increasing cAMP hydrolysis, Mironid®’s first-in-class small molecule LoAc® compounds [1] directly target the increased kidney cAMP levels that are known to drive cyst formation in ADPKD, resulting in decreased cyst growth in vitro and in vivo. Here we present translational data demonstrating that LoAc® compound MR-L22 lowers renal cAMP after single or multiple doses and delivers efficacy in the Pkd1RC/RC mouse model of ADPKD, alongside favourable differentiation from tolvaptan, the only therapy approved for ADPKD patients. Method To demonstrate cAMP lowering in urine, male Han Wistar rats (n = 6) were dosed p.o. with 100 mg/kg MR-L22 or vehicle (1% methylcellulose, 0.2% Tween 80) either once, or on 7 consecutive days. Urine was collected on Day 1 or Day 7. To assess the ability of MR-L22 to ameliorate disease progression alone, or in combination with tolvaptan, doses of MR-L22 (60 mg/kg) and tolvaptan (0.1% in chow), both expected to be individually sub-maximally efficacious, were administered singly, or in combination in F1 hybrid progeny Pkd1RC/RC mice (8 m and 8f/group), from 4-16 weeks of age. Urine measurements were taken after 8 weeks of dosing. Total kidney volume (TKV) was measured by MRI prior to randomisation and termination. cAMP in urine and kidney tissue were measured by ELISA. Results A 100 mg/kg oral dose of MR-L22 in rat resulted in a &amp;gt;40% decrease in cAMP in urine compared to vehicle. This effect was maintained over multiple days of dosing demonstrating that urinary cAMP can be used as a biomarker of LoAc® target engagement. Consistent with the mechanism of action, a small increase in urine production and commensurate decrease in urine osmolality was observed. To assess the ability of MR-L22 to ameliorate disease progression in an in vivo model of ADPKD, and to determine whether additive or synergistic effects might be seen in combination with tolvaptan, MR-L22 (60 mg/kg) and tolvaptan (0.1% in chow) were dosed individually and in combination in the Pkd1RC/RC mouse model of ADPKD. Alongside a reduction in urinary cAMP, MR-L22 delivered reductions in TKV/Bw (Fig. 1), urinary and tissue cAMP, cystic index, terminal blood urea nitrogen (BUN) and creatinine, as well as trending reductions in urinary NGAL. Conclusion We have shown that in rat, acute or multiple dosing, of LoAc® compound MR-L22 results in a significant reduction of urinary cAMP and a small, but measurable, increase in urine production and commensurate decrease in urine osmolality. Furthermore, we have demonstrated that significantly reduced urinary and kidney cAMP levels correlate with reductions in TKV, cystic index, Kw/Bw, BUN and urinary NGAL in the Pkd1RC/RC model of ADPKD. In these studies, we have demonstrated beneficial differentiation vs tolvaptan on limiting aquaretic effects but been unable to conclusively demonstrate additive or synergistic effects of MR-L22 combined with tolvaptan due to experimental limitations; Further experiments will be needed to answer this question. Looking forwards, measurements of urine cAMP and urine osmolality will be measured as supportive of target engagement in phase 1 studies. Positive effects on TKV, seen in vivo in the Pkd1RC/RC model, will be assessed in a phase 2 clinical trial. By directly targeting cAMP, LoAc® compounds have the potential to slow renal disease progression, and to delay or prevent the need for dialysis, offering a potential new treatment option for ADPKD patients.

Developing a Clinically Relevant Hemorrhagic Shock Model in Rats.

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic shock, i.e., organ failure due to rapid loss of a large volume of blood, is associated with a highly complex pathophysiology involving several pathways. Numerous existing animal models of hemorrhagic shock strive to replicate what happens in humans, but these models have limits in terms of clinical relevance, reproducibility, or standardization. The aim of this study was to refine these models to develop a new model of hemorrhagic shock. Briefly, hemorrhagic shock was induced in male Wistar Han rats (11-13 weeks old) by a controlled exsanguination responsible for a drop in the mean arterial pressure. The next phase of 75 min was to maintain a low mean arterial blood pressure, between 32 mmHg and 38 mmHg, to trigger the pathophysiological pathways of hemorrhagic shock. The final phase of the protocol mimicked patient care with an administration of intravenous fluids, Ringer Lactate solution, to elevate the blood pressure. Lactate and behavioral scores were assessed 16 h after the protocol started, while hemodynamics parameters and plasmatic markers were evaluated 24 h after injury. Twenty-four hours post-hemorrhagic shock induction, the mean arterial and diastolic blood pressure were decreased in the hemorrhagic shock group (p< 0.05). Heart rate and systolic blood pressure remained unchanged. All organ damage markers were increased with the hemorrhagic shock (p < 0.05). The lactatemia and behavioral scores were increased compared to the sham group (p < 0.05). In conclusion, we demonstrated that the protocol described here is a relevant model of hemorrhagic shock that can be used in subsequent studies, particularly to evaluate the therapeutic potential of new molecules.

Експериментальне дослідження гепатопротекторної дії комплексу n-оксид-2,6-диметил піридину з бурштиновою кислотою (Потейтіну) на моделі токсичного гепатиту, спричиненого тетрахлорметаном

Plant growth regulators (PGR), in particular, methyl derivatives of pyridine N-oxide, reduce the acute toxicity of pesticides, reduce the hepatotoxic effects of chlorpyrifos and ease the course of intoxication under long-term exposure. The role of the body’s defence systems under the combined effects of PGR and pesticides has not yet been sufficiently elucidated. The study of the protective effect of PGR Poteitin under the conditions of toxic damage to the liver by carbon tetrachloride (CCl4) will allow finding out the mechanisms of its combined action. The results of the research will be used to develop preventive measures for pesticide poisoning. Aim. Investigate the hepatoprotective properties of the 2.6-dimethylpyridine N-oxide complex with succinic acid (Poteitin) on the model of acute hepatitis caused by carbon tetrachloride. Materials and Methods. Acute hepatitis was modelled on male Wistar Han rats with subcutaneous injection of CCl4 (pure substance) in petroleum jelly oil (0.8 ml/100 g of body weight) for 2 days. Poteitin was administered at the doses of 23 and 0.23 mg/kg, reference substance Silybor 35 – at the dose of 5 mg/kg. Intact rats received petroleum jelly oil in the same volumes. Poteitin and Silybor were administered in a treatment-prophylactically regimen, orally 1 hour pre- and 2 hours post-CCl4. The activity of enzymes of cytolysis and cholestasis, indicators reflecting liver function, the content of lipid peroxidation (LP) products and antioxidant activity were determined in blood serum. Histological studies of liver tissue were carried out. The index of effectiveness of the protective effect of Poteitin was calculated The results were statistically processed using the one-way ANOVA followed by Fisher's LSD post-hoc test. Results and Discussion. CCl4 caused acute liver damage: increased activity of markers of cytolysis and cholestasis, decrease in a number of functional tests, activity of catalase, LP products accumulation, fatty and balloon dystrophy of hepatocytes, lympho-leukocyte infiltration. Poteitin on the background of CCl4 contributed to the normalization of the activity of markers of cytolysis and cholestasis, the restoration of functional indicators, reduced the content of LP products, increased the activity of catalase, and reduced liver damage. The protective effect of Poteitin was not inferior to Silybor. Conclusions. 1. Under the conditions of acute hepatitis, carbon tetrachloride causes moderate liver damage. 2. Poteitin at the doses of 23 and 0.23 mg/kg has a pronounced hepatoprotective effect: it normalizes the activity of enzymes of cytolysis and cholestasis, functional indicators – the level of glucose, cholesterol, total protein and bilirubin, urea; reduces the intensity of LP and activation of the antioxidant system. 3. The protective effect of Poteitin at both tested doses as cytolysis and cholestasis indicators, functional tests and morphostructural changes in the liver is comparable to the reference drug Silybor. The protective effect of Poteitin, determined by the intensity of LP, is greater than that of Silybor. Keywords: acute hepatitis, carbon tetrachloride, hepatoprotective effect, 2.6-dimethylpyridine N-oxide complex with succinic acid.

Hepatoprotective effect of 2,6-dimethylpyridine N-oxide (Ivin) in experimental model of CCl(4)-induced hepatitis of rats

The effect of concomitant pesticides and plant growth regulators on humans is still not well understood. N-oxide-2,6-dimethylpyridine (Ivin) is the plant growth regulator known to reduce the acute toxicity of pesticides, but its protective mechanisms need to be investigated. The aim of the study was to assess the hepatoprotective ability of 2,6-dimethylpyridine N-oxide (Ivin) using a model of acute CCl4-induced hepatitis. Male Wistar Han rats received two subcutaneous CCl4 injections (0.8 ml/100 g b.w.). Oral Ivin (13 or 0.13 mg/kg) and hepatoprotector “Silybor-35” (5 mg/kg) as reference substances were administered orally one hour pre- and 2 hours post-CCl4 injection. The biochemical assay of blood plasma, estimation of lipid peroxidation products in the liver tissue and histological liver analysis were done. The results of functional tests and histomorphological studies of liver tissue demonstrated that Ivin exhibited a pronounced hepatoprotective effect, more pronounced when it was administered at a low 0.13 mg/kg dose. Calculation of the hepatoprotection efficiency index for Ivin showed that it was comparable to that for “Silybor-35”. Keywords: hepatoprotective effect, Ivin, СCl4-induced hepatitis of rats

The Interaction of Dietary Pectin, Inulin, and Psyllium with Copper Nanoparticle Induced Changes to the Cardiovascular System.

We aimed to analyze how supplementation with a standard (recommended, 6.5 mg/kg) or enhanced (two-times higher, 13 mg/kg) dose of copper (Cu), in the form of nanoparticles (NPs) along with dietary intervention via the implementation of diverse types of fiber, affects the cardiovascular system in rats. Nine-week-old male Wistar Han rats (n/group = 10) received, for an additional 6 weeks, a controlled diet with cellulose as dietary fiber and ionic Cu (in the form of carbonate salt). The experimental groups received cellulose, pectin, inulin, and psyllium as dietary fiber, together with CuNPs (6.5 or 13 mg/kg diet). After the experimental feeding, samples of blood, hearts, and thoracic arteries were collected for further analysis. Compared to pectin, and under a standard dose of CuNPs, inulin and psyllium beneficially increased the antioxidant capacity of lipid- and water-soluble compounds in the blood, and decreased heart malondialdehyde. Moreover, pectin decreased heart catalase (CAT) and cyclooxygenase (COX)-2 in the aortic rings compared to inulin and psyllium under standard and enhanced doses of copper. When the dose of CuNPs was enhanced, inulin and psyllium potentiated vasodilation to acetylcholine by up-regulation of COX-2-derived vasodilator prostanoids compared to both cellulose and pectin, and this was modulated with selective inducible nitric oxide synthase (iNOS) inhibitor for psyllium only. Moreover, inulin decreased heart CAT compared to psyllium. Our results suggest that supplementation with dietary fiber may protect the vascular system against potentially harmful metal NPs by modulating the antioxidant mechanisms.

Стан прооксидантної та антиоксидантної систем організму щурів за умов однократної дії деяких метильних похідних N-оксид піридину – Івіну і Потейтіну

АBSTRACT. Previously, it was shown that pyridine N-oxide based plant growth regulators (PGR) Ivin and Poteitin when combined with pesticides reduce the acute and subchronic toxicity of pesticides for mammals, have a hepatoprotective effect, contribute to the reduction of mutagenic activity, in particular, of Cyclophosphane and Dioxidin cytostatics (Vasetska O.P., 2017, Vasetska O.P. et al., 2020, 2021). It is possible that a wide range of their biological effects may be associated with the activation of the body’s protective systems, in particular the antioxidant system. The effect of these PGRs on the state of the pro-oxidant and antioxidant systems is not sufficiently clarified. Therefore, an important issue for preventive toxicology is the study of the nature of the effect of PGR on the state of the body’s pro-oxidant and antioxidant systems, which will contribute to the development of preventive measures to preclude acute and chronic intoxications with pesticides. Aim. To find out the state of the body’s pro-oxidant and antioxidant systems under a single oral exposure to some plant growth regulators –pyridine N-oxide methyl derivatives. Materials and Methods. For the aim of this research we used PGR 2.6-dimethyl- pyridine N-oxide (Ivin) and a complex of 2.6- dimethylpyridine-N-oxide with succinic acid (Poteitin). PGR in the form of an aqueous solution was administered to male Wistar Han rats orally with a probe: Ivin at doses of 650 mg/kg (1/2 LD50) and 13 mg/kg (1/100 LD50), Poteitin – at doses of 1150 mg/kg (1/2 LD50) and 23 mg/kg (1/100 LD50). The influence of Ivin and Poteitin on the lipid peroxidation (LPO) and the antioxidant system under a single oral exposure to the body of male rats was determined on days 1, 3, and 7. LPO in liver tissues was determined by the content of malondialdehyde (MDA), diene conjugates (DC), ketodienes (KD), conjugated trienes (CT) and Schiff bases (SB). MDA level was determined by the reaction with 2-thiobarbituric acid, DC, KD, CT, and SB in liver tissues – by the extraction-spectrophotometric method. The antioxidant status of the body was assessed by the activity of the catalase enzymes (CAT), glutathione peroxidase (GP), glutathione reductase (GR), the content of reduced glutathione (GSH) and total antioxidant activity (AOA). The results of the research were estimated by standard methods of variational statistics according to Student’s t-test criterion. Results. It was established that Ivin in the researched doses in the heptane fraction reduces the LPO intensity (containing MDA, DC, SB) in the liver tissues of rats, in the isopropanol fraction – DC; increases the activity of catalase and total AOA in blood serum. Poteitin in the tested doses in the heptane fraction reduces the formation of lipid products (containing MDA, DC, KD and CT) in the liver tissues of rats, in the isopropanol fraction – DC; increases the activity of catalase and the content of GSH, total AOA. Both substances do not disrupt the glutathione system. Compared to Ivin, Poteitin has a more pronounced effect on the intensity of LPO and the activity of the antioxidant system. Conclusions. 1. Plant growth regulators Ivin and Poteitin, at a single exposure to the body of male rats in doses corresponding to 1/2 and 1/100 LD50, reduce pro-oxidant activity, do not disrupt the glutathione system, increase the activity of the antioxidant system. 2. Increase in the activity of the antioxidant system and inhibition of the intensity of lipid peroxidation processes both under the influence of Ivin and Poteitin are aimed at adapting the body to the chemical factor. 3. Activation of the antioxidant system together with a decrease in the intensity of LPO can be one of the mechanisms of protection of the body from the toxic effects of pesticides when they are simultaneously introduced into the body with plant growth regulators based on methyl derivatives of pyridine N-oxide. Key words: plant growth regulators, Ivin, Poteitin, prooxidant and antioxidant systems.

#6489 UNI-494 DOES NOT SIGNIFICANTLY AFFECT RAT RESPIRATORY FUNCTION

Abstract Background and Aims Nicorandil, a selective mitochondrial ATP-sensitive potassium channel activator, may be beneficial for several disease states, including acute kidney injury. However, its clinical use is limited by serious gastrointestinal side effects and rapid absorption and elimination. UNI-494, a novel nicorandil prodrug designed to improve its pharmacologic properties, may increase the short half-life and improve the safety profile of nicorandil. We present safety results from a safety pharmacology study evaluating the effects of UNI-494 on respiratory function in a rat model. Method The effects of UNI-494 on respiratory function (peak inspiratory and expiratory flow, inspiratory and expiratory time, minute volume, and respiratory rate) were evaluated using whole body plethysmography test in 4 groups of male Wistar Han rats (n = 8 per group). In addition to a control group, 3 test groups were administered doses of 2, 20, or 200 mg UNI-494/kg. Airway function parameter data generated were acquired using specialized EMKA Technologies software (IOX version 2.9.5; DATANLYST version 2.6.1) connected to Emka plethysmographic equipment. Recordings were taken 3 hours before and 4 hours after administration of the control and test items. Effects were reported at 0, 10, 15, 20, 30, 60, 90, 120, 180, and 240 minutes after administration. Results UNI-494 had no relevant effects on rat respiratory function over the 240-minute test period at any dose (2, 20, or 200 mg UNI-494/kg). There were small but insignificant differences in peak inspiratory and expiratory flow (Figure 1, 2), minute volume, respiratory rate, and inspiratory and expiratory time between the control group and the 200 mg UNI-494/kg group. Conclusion Rat respiratory function was not significantly affected by UNI-494. Future studies should continue to evaluate the safety and efficacy of this novel drug.

Sex-related differences in retinal function in Wistar rats: implications for toxicity and safety studies.

Introduction: Wistar Han rats are a preferred strain of rodents for general toxicology and safety pharmacology studies in drug development. In some of these studies, visual functional tests that assess for retinal toxicity are included as an additional endpoint. Although the influence of gender on human retinal function has been documented for more than 6decades, preclinically it is still uncertain if there are differences in retinal function between naïve male and female Wistar Han rats. Methods: In this study, sex-related differences in the retinal function were quantified by analyzing electroretinography (ERG) in 7-9-week-old (n = 52 males and 51 females) and 21-23-week-old Wistar Han rats (n = 48 males and 51 females). Optokinetic tracking response, brainstem auditory evoked potential, ultrasonic vocalization and histology were tested and evaluated in a subset of animals to investigate the potential compensation mechanisms of spontaneous blindness. Results/Discussion: Absence of scotopic and photopic ERG responses was found in 13% of 7-9-week-old (7/52) and 19% of 21-23-week-old males (9/48), but none of female rats (0/51). The averaged amplitudes of rod- and cone-mediated ERG b-wave responses obtained from males were significantly smaller than the amplitudes of the same responses from age-matched females (-43% and -26%, respectively) at 7-9weeks of age. There was no difference in the retinal and brain morphology, brainstem auditory responses, or ultrasonic vocalizations between the animals with normal and abnormal ERGs at 21-23weeks of age. In summary, male Wistar Han rats had altered retinal responses, including a complete lack of responses to test flash stimuli (i.e., blindness), when compared with female rats at 7-9 and 21-23weeks of age. Therefore, sex differences should be considered when using Wistar Han rats in toxicity and safety pharmacology studies with regards to data interpretation of retinal functional assessments.

The small molecule ACM-001 improves cardiac function in a rat model of severe cancer cachexia.

Cachexia, a common manifestation of malignant cancer, is not only associated with weight loss, but also with severe cardiac atrophy and impaired cardiac function. Here, we investigated the effects of ACM-001 (0.3 or 3mg/kg/d) in comparison to carvedilol (3 or 30mg/kg/d), metropolol (50 or 100mg/kg/d), nebivolol (1 or 10mg/kg/d) and tertatolol (0.5 or 5 mg/kg/d) on cardiac mass and function in a rat cancer cachexia model. Young male Wistar Han rats were inoculated with 108 Yoshida hepatoma AH-130 cells ip and treated once daily with verum or placebo by gavage. Cardiac function (echocardiography), body weight and body composition (NMR-Scans) were assessed. The hearts of animals were euthanized on day 11 (placebo and 3 mg/kg/d ACM-001) were used for signaling studies. Beta-blockers had no effect on tumour burden. ACM-001 reduced body weight loss (placebo: -34±2.4g vs 3mg/kg/d ACM-001: -14.8±8.4g, p=0.033). Lean mass wasting was attenuated (placebo: -16.5±2.34g vs 3mg/kg/d ACM-001: -2.4±6.7g, p=0.037), while fat loss was similar (p=0.4) on day 11. Placebo animals lost LVmass (-101±14mg), which was prevented only by 3mg/kg/d ACM-001 (7±25mg, 0<0.01 vs placebo). ACM-001 improved the EF (ΔEF: placebo: -24.3±2.6, 3mg/kg/d ACM-001: 0.1±2.9, p<0.001). Cardiac output was 50% lower in the placebo group (-41±4mL/min) compared to baseline, while 3mg/kg/d ACM-001 preserved cardiac output (-5±8, p<0.01). The molecular mechanisms involve inhibition of protein degradation and activation of protein synthesis pathways. This study shows that 3mg/kg/d ACM-001 restores the anabolic/catabolic balance in cardiac muscle leading to improved function. Moreover, not all beta-blockers have similar effects. This article is protected by copyright. All rights reserved.

Функціональний стан печінки за одноразового перорального впливу на організм деяких метильних похідних n-оксид піридину

RESUME. Pesticides occupy one of the priority places among environmental pollutants. The hepatobiliary system disorder is a characteristic of most pesticides. The hepatotoxic effect of synthetic plant growth regulators (PGRs) based on pyridine N-oxide isn’t explored enough. The hepatotoxic effect PGR nature finding is an important aspect of preventive toxicology. Aim. To study the effect of some pyridine-N-oxide methyl derivatives on the liver functional state under a single oral dosage into the body. Materials and Methods. For the research were used PGRs – 2.6-dimethylpyridine-N-oxide (Ivin), 99.9 % and complex 2.6- dimethylpyridine-N-oxide with succinic acid (Poteitin), 99.9 %. During the experiment which was carried out on male Wistar Han rats, it was a single oral administration of aqueous solution form of test substances: Ivin in doses of 650 and 13 mg/kg and Poteitin in doses 1150 and 23 mg/kg. The activity of the alanineaminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (ALP), the content of total proteins, glucose, triglycerides, cholesterol, urea and creatinine in the blood serum were determined on the 1st, 3rd, and 7th day using “Filisit- Diagnostics” reagent kit. EPR signals (g-2.25; g-2.14; g-2.05; g-2.03; g-2.00; g-1.97; g-1.94) were determined in liver tissue on the radio spectrophotometer “Varian E-109” (USA) at a temperature of 77 K. The results. Ivin at a dose of 13 mg/kg in particular study periods significantly increased the content of urea and glucose. At a dose of 650 mg/kg, the activity of AST, the content of urea, creatinine, and glucose significantly increased, and the activity of ALP decreased. Poteitin in a dose of 23 mg/kg in particular study times significantly increased the content of urea and creatinine, and the activity of ALP in the blood serum. At a dose of 1150 mg/kg, a significant decrease in the activity of ALP, and an increase in the content of urea, creatinine and glucose in the blood serum were revealed. The majority of liver EPR signals were reduced due to the influence of Ivin and Poteitin in both studied doses in different terms. Ivin at a dose of 13 mg/kg decreased the content of Mo7+-containing proteins and free radicals (FR) on 1st and 7th days respectively. At a dose of 650 mg/kg increased the content of iron-sulfur proteins (ISP) in 1stday of experiment and there was a decrease in the content of cytochrome P-450 (CytP-450), nitrosyl complexes, ISP and free radicals (FR) in 7 days. Poteitin at a dose of 23 mg/kg decreased the content of cyt-P-450, FR, ISP, and Mo7+–containing proteins, and increased the content of Cu2+-containing proteins. At a dose of 1150 mg/kg, there was a decrease in the content of cyt-P-450, BP, and ISP (for 1st and 3th days) and their increase for 7 days. Conclusions. With acute oral exposure, Ivin and Poteitin show a weak hepatotropic effect, characterized by a hyperglycemia, a slight decrease in the activity of alkaline phosphatase and an increase in urea and creatinine levels in the blood serum. Hyperglycemia can be associated with a stressogenic reaction to a chemical factor. This may indicate a carbohydrate metabolism disorders and various pathological conditions with long-term exposure to the studied substances. Intensification of the protein degradation and the accumulation of urea and creatinine in the blood serum can lead to a disorder of the filtering and excretory functions of the kidneys, which requires further research and clarification. Ivin and Poteitin reduce the EPR spectra of rat liver – cyt-P-450, free radicals, iron-sulfur proteins, nitrosyl complexes of non-heme iron, which indicates a decrease in the activity of the monooxygenase system, inhibition of oxidation processes, the formation disruption of iron-sulfur complexes and a decrease in the labile nitrosyl complex formation. A decrease in the content of Mo7+-containing proteins and an increase in the content of Cu2+-containing proteins may be associated with the activation of antiradical processes in the liver of rats. Key Words: plant growth regulators, Ivin, Poteitin, hepatotoxic effect, liver EPR spectra.

Dietary Effects of Chromium Picolinate and Chromium Nanoparticles in Wistar Rats Fed with a High-Fat, Low-Fiber Diet: The Role of Fat Normalization

We aimed to evaluate how feeding a high-fat-low-fiber (F) diet to rats and dietary intervention with the implementation of a standard-fat-and-fiber (S) diet affects the response of the cardiovascular system to chromium (III) picolinate (Cr-Pic) and, alternatively, chromium nanoparticles (Cr-NPs). Young male Wistar Han rats (n/group = 12) from either the fatty group (18 weeks on F diet) or the intervention group (9 weeks on F diet + 9 weeks on S diet) received a pharmacologically relevant dose of 0.3 mg Cr/kg body weight in the form of Cr-Pic or Cr-NPs for 9 weeks. Our study on rats confirmed the pro-inflammatory effect of an F diet administered for 18 weeks. In the intervention group, both Cr-Pic and Cr-NPs decreased heart glutathione ratio (GSH+GSSG), enhanced participation of nitric oxide (NO) derived from inducible NO synthase (iNOS) in vascular relaxation to acetylcholine (ACh), increased the vasodilator net effect of cyclooxygenase-2 (COX-2)-derived prostanoids, and increased the production of superoxide anion (O2.-) in aortic rings. Meanwhile, in the fatty group, there was increased heart superoxide dismutase (SOD), decreased heart catalase (CAT), and reduced sensitivity in pre-incubated aortic rings to endogenous prostacyclin (PGI2). The factors that significantly differentiated Cr-NPs from Cr-Pic were (i) decreased blood antioxidant capacity of water-soluble compounds (0.75-fold, p = 0.0205), (ii) increased hydrogen peroxide (H2O2) production (1.59-fold, p = 0.0332), and (iii) modified vasodilator response due to PGI2 synthesis inhibition (in the intervention group) vs. modified ACh-induced vasodilator response due to (iv) COX inhibition and v) PGI2 synthesis inhibition with thromboxane receptor blockage after 18 weeks on F diet (in the fatty group). Our results show that supplementation with Cr-Pic rather than with Cr-NPs is more beneficial in rats who regularly consumed an F diet (e.g., for 18 weeks). On the contrary, in the intervention group (9 weeks on F diet + 9 weeks of dietary fat normalization (the S diet)), Cr-Pic and Cr-NPs could function as pro-oxidant agents, initiating free-radical reactions that led to oxidative stress.

Introduction of gloved hand to cage induces 22-kHz ultrasonic vocalizations in male albino rats.

Rodents emit ultrasonic vocalizations (USVs) above the human hearing threshold of ~ 20 kHz to communicate emotional states and to coordinate their social interactive behavior. Twenty-two kHz USVs emitted by adult rats have been reported in a variety of aversive social and behavioral situations. They occur not only under painful or restraining conditions but can also be evoked by gentle cutaneous touch or airflow. This study aimed to test if placement of a human hand in a cage can evoke 22-kHz USVs. It was found that 36% of the adult male Sprague-Dawley and 13% of the adult male Wistar Han rats emitted 22-kHz USVs when a gloved hand was introduced into the cages. Average vocalization onset latencies were 5.0 ± 4.4 s (Sprague-Dawley) and 7.4 ± 4.0 s (Wistar Han) and the USVs had a stable frequency (22 kHz) across the calls, ranging from 0.1 to 2.3 seconds in duration. Surprisingly, no 22-kHz USVs were found in any female Wistar Han rats tested. To further explore the mechanisms underlying this observation, we compared retinal function, basal serum corticosterone, and testosterone levels between the 22-kHz USV responders and non-responders. None of these parameters or endpoints showed any significant differences between the two cohorts. The results suggest that the introduction of a gloved-hand inside the cage can trigger adult male albino rats to emit 22-kHz ultrasonic vocalizations. This response should be considered in USV studies and animal welfare.

Changes in the Behavior and Body Weight of Mature, Adult Male Wistar Han Rats after Reduced Social Grouping and Social Isolation.

Changes in housing density, including individual housing, are commonly necessary in animal research. Obtaining reproducibility and translational validity in biomedical research requires an understanding of how animals adapt to changes in housing density. Existing literature mainly addresses acclimatization after transportation. We used a within-subject design to examine changes in behavior and weight gain of 4-mo-old male Wistar Han rats after reduction of their social group (RSG; due to removal of one rat from a cage containing 3 rats) and social isolation (SI; the removed rat) for the subsequent 2 wk. Changes in weight gain and in exploratory and center-avoidance behavior in an inescapable open arena (OA) were measured before (D0) and on days 7 and 14 (D7 and D14, respectively) after social change. The motor response to d-amphetamine (1.5 mg/kg), which stimulates behavioral arousal in response to novelty, was assessed at D14. Within-subject design revealed that RSG rats in OA had less locomotion at D7 but not more center-avoidance behavior and had returned to the D0 activity level at D14; SI rats in OA had consistently less locomotion and more center-avoidance behavior. Rearing behavior during OA exposure did not change in either group. However, SI rats showed more center-avoidance behavior in OA, greater weight gain, and less amphetamine-induced rearing at D14 as compared with RSG rats. These data indicate that after RSG, mature adult male rats require 2 wk to return to their baseline level of OA-related behavior, while after SI they gain weight and acquire maladaptive exploratory and center-avoidance behavior. The finding that SI produces maladaptive behavioral and physiologic alterations in adult male rats deserves attention because these changes could have confounding effects on research findings.

N-acetylcysteine improves impulse control and attenuates relapse-like alcohol intake in long-term drinking rats

Increasing evidence suggests that individuals with alcohol use disorder (AUD) present with a disrupted glutamatergic system that underlies core components of addictive disorders, including drug relapse and low impulse control. N-acetylcysteine (NAC) is a cystine prodrug that has been found to promote glutamate homeostasis and drug abstinence. However, no studies to date have evaluated NAC’s effect on impulsivity in substance use disorders. Here we determined whether NAC would decrease alcohol-intake behaviors, in addition to improving impulse control, in long-term alcohol drinking male Wistar-Han rats. Before the start of the experiments, all rats were exposed to long-term intermittent access to 20% ethanol for at least seven weeks. Next, in different groups of rats, the effect of NAC (60 and/or 90 mg/kg) was evaluated on (i) voluntary alcohol drinking using a two-bottle free choice paradigm, (ii) the motivation to self-administer alcohol under a progressive ratio schedule of reinforcement, and (iii) relapse-like drinking using the alcohol deprivation effect model. Finally, (iv) NAC’s effect on impulse control was evaluated using the five-choice serial reaction time task. Results showed that NAC administration at 90 mg/kg significantly reduced relapse-like drinking and improved impulse control. In contrast, NAC had no effect on levels of alcohol drinking or motivation to drink alcohol. In conclusion, our findings continue to support the use of NAC as an adjuvant treatment for the maintenance of abstinence in AUD. Moreover, we provide evidence for NAC’s efficacy in improving impulse control following drinking, which warrants further investigation in substance use settings.

Effect of the analgesics dipyrone, tramadol, and meloxicam on the behavior of laboratory rats

Awareness about the ethical use of laboratory animals has increased over recent decades alongside the development of strict guidelines and legislation concerning the welfare of animals in scientific research. Although minimization of animal suffering during experimentation is of paramount importance, it is sometimes neglected because of the presumed interference of analgesics on behavioral patterns. For this reason, comprehensive research with multiple pharmacological agents is vital if analgesics are to be included in experimental procedures. In this context, we aimed to evaluate the effects of the analgesics dipyrone (DIP), tramadol (TRA), meloxicam (MEL), and combinations thereof on the behavioral parameters of naïve laboratory rats. Forty-eight SPF male Wistar-Han rats were divided randomly into 6 equal groups (n = 8 per group) and treated by intraperitoneal injection with DI (178 mg/kg), TRA (17.8 mg/kg), MEL (1.5 mg/kg), DIP+TRA (178 + 17.8 mg/kg), MEL+TRA (1.5 + 17.8 mg/kg) or saline (SAL; control). The behavior of the treated animals was assessed 30 minutes after injection of analgesic by sequential submission to Open Field, Elevated Plus Maze, and Grooming Transfer tests. Treatment with DIP+TRA provoked the most significant alterations in rat behavior by reducing locomotion, rearing, and grooming. Although treatment with DI alone diminished exploratory behavior, the effect on grooming was not remarkable, thereby suggesting that DI has a suppressive motor effect, possibly caused by its action on the endocannabinoid system, which is potentiated by TRA. In the Open Field tests, the grooming behavior of all TRA-treated animals was reduced while treatment with MEL increased locomotion. Given that analgesia is an ethical duty, the experimental design should always consider the behavioral effects of the drugs themselves.

Sucrose intake and preference by Wistar Han rats are not influenced by sex or food/water deprivation

Anhedonia is the decreased ability to experience pleasure from rewarding or enjoyable activities, a core symptom of depression. The sucrose preference test (SPT), based on a two-bottle choice paradigm, is a widely used behavioural paradigm for the evaluation of anhedonia in rodents. Up to now, different protocols have been reported regarding water/food deprivation and duration of exposure to the water/sucrose solutions. In this work, by comparing six of the most used SPT protocols regarding sucrose preference and total intake, in both male and female Wistar Han rats, we showed (i) food/water deprivation does not significantly impact sucrose intake and preference; (ii) increasing the duration of the test is associated with an increased sucrose preference and (iii) no sex-specific differences in the basal sucrose preference of Wistar Han rats. Our results call for standardization of protocols and suggest a protocol without food/water deprivation and a 12-hour duration (lights out) as more efficacious in the measurement of anhedonia in rodents. This protocol not only reduces the confounding factors of drinking patterns and the stress-inducing food/water deprivation but also is not sensitive to sex-specific differences in the total intake of liquid in Wistar Han rats.

Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species

Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as day 8 in rats (≥5 × 1013 vg/kg) and day 14 in monkeys (≥3.3 × 1013 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species.

Comparative analysis of two methodological approaches to the study of endocrine disruptor alpha-cypermethrin reproductive toxicity

At present, one of the main threats to humanity is undoubtedly endocrine disruptors (ED), since they directly disrupt the processes of ensuring homeostasis, which are identical to the very essence of the concept of health, considered in valeology as the ability to maintain age-appropriate stability in conditions of sharp changes in quantitative and qualitative parameters of the triune flow of sensory, verbal and structural information. Pesticides can disrupt the physiological functioning of many endocrine chains, including the endocrine mechanisms that ensure reproductive health. The study aimed to compare the results of our studies of alpha-cypermethrin reproductive toxicity in the test system for studying gonadotoxic activity with data obtained in the test system "Three Generation Reproduction Study". The studies were performed on male and female Wistar Han rats with two generic samples of alpha-cypermethrin from different manufacturers at doses of 0.2, 1.0 and 3.0 mg/kg body weight. The exposure lasted 11 (males) and 10 (females) weeks. During the last two weeks of exposure the state of the estrous cycle, duration and frequency of each stage in females was studied. After the end of treatment functional parameters of the state of the gonads in males and the ability of animals to reproduce were examined. In males, the total sperm count, the absolute and relative number of motile germ cells, and the number of pathologically altered forms were evaluated. The results of the study showed that exposure of male and female Wistar Han rats to alpha-cypermethrin at doses of 0.2–3.0 mg/kg during gametogenesis had a toxic effect on the reproductive system characterized by impaired gonadal and reproductive functions. In our studies, alpha-cypermethrin was found to have reproductive toxicity (reduced number of corpora luteum and live fetuses, increased absolute and relative postimplantation death, reduced average weight of fetuses and litters) and endocrine-disruptive effect, having a pronounced antiandrogenic effect on males. Obvious signs of endocrine reproductive disorders (changes in testis and epididymis weight, deterioration in semen parameters, altered length of separate stages of the estrous cycle) were observed in both females and males. However, studies in a test system of three-generations did not reveal a reproductive and endocrine-disruptive effect of cypermethrin, the toxicity of which was recognized as equivalent to the toxicity of alpha-cypermethrin. The obtained results showed higher sensitivity, informative and diagnostic significance of the methods for studying gonadotoxicity than the methodology of reproductive toxicity studies in the test system “Three Generation Reproduction Study”.

Bioaccessibility as a determining factor in the bioavailability and toxicokinetics of cadmium compounds

Cadmium toxicity occurs where there is absorption and accumulation of cadmium ions (Cd2+) in tissues beyond tolerable levels. Significant differences in the release of Cd2+ from cadmium compounds in biological fluids, like gastric fluid, may indicate differences in bioavailability and absorption. This means that direct read-across from high solubility cadmium compounds to lower solubility compounds may not accurately reflect potential hazards. Here, the relative bioaccessibility in gastric fluid of cadmium telluride and cadmium chloride was evaluated using in vitro bioelution tests whilst the toxicokinetic behavior of these two compounds were compared after dietary administration for 90 days in male and female Wistar Han rats following OECD TG 408. Cadmium chloride was highly bioaccessible, whilst cadmium telluride showed low solubility in simulated gastric fluid (90 % and 1.5 % bioaccessibility, respectively). This difference in bioaccessibility was also reflected by a difference in bioavailability as shown by the difference in the liver and kidney concentrations of cadmium after repeat oral exposure. Feeding at doses of 750 and 1500 ppm of cadmium telluride did not result in tissue cadmium levels above the lower limit of quantification (LLOQ). In contrast, feeding with a lower test substance concentration yet higher concentration of bioaccessible cadmium (30 ppm cadmium chloride) resulted in tissue accumulation of cadmium. Only slight, non-adverse changes in hematology and clinical chemistry parameters were seen at these doses, indicating an absence of significant cadmium mediated toxicity towards target organs (kidney and liver), reflected in minimal cadmium accumulation in these organs.This study demonstrates that bioelution tests can help determine the bioaccessibility of cadmium, which can be used to estimate the potential for target tissue toxicity based on known toxicokinetic profiles and threshold levels for cadmium toxicity, while reducing and refining animal testing.