Hepatocellular Hypertrophy Research Articles

Effects of urban streams on muscle non-protein thiols, gill and liver histopathology in zebrafish (Danio rerio) assessed by active biomonitoring

Aquatic biota are exposed to toxic substances resulting from human activities, reducing environmental quality and can compromise the health of the organisms. This study aimed to employ Danio rerio as an environmental bioindicator, analyzing the effects of water from distinct urban aquatic environments. An active biomonitoring system was set up to compare the temporal dynamics of histological biomarkers for gill and liver and the patterns of non-protein thiols (NPSH) in muscle in specimens exposed for 3, 6, and 12 days. Three large urban basins in the city of Campo Grande (Midwest of Brazil) were selected. Two sites are in a very populous area (i.e Lagoa and Bandeira) and another on in an area with agricultural activities (i.e Anhanduí). All the streams displayed distinct qualitative characteristics. The presence of metals, including Mn, Zn, Fe, and Al, as well as pH, temperature, and dissolved oxygen, accounted for 38% of the variability (PC1), while total solids, conductivity, ammonia, nitrite, and explained 24 % (PC2). Degree tissue changes index (DTC) in gill and the concentration of NPSH increased in all streams during 3, 6 and 12 days of exposure. DTC in liver increases in all exposure times in most populous stream (i.e Lagoa and Bandeira). Histopathological evidence in the gill, including proliferation, desquamation, and necrosis of the primary lamellar epithelium; fusion and aneurysms in the secondary lamellar epithelium were observed after three days of exposure. Degenerative nuclear figures were noted in the liver after three days of exposure, followed by hepatocellular hypertrophy, lipidosis, and necrosis at twelve days. Our findings showing time-dependent effects of urban aquatic environments in histopathological (i.e DTC) and biochemical biomarkers in zebrafish. The biomonitoring model enabled a comparison of the temporal dynamics of various health markers, using zebrafish as bioindicator. Future studies might use this experimental model and biomarkers for environmental biomonitoring program.

General toxicity and screening of reproductive and developmental toxicity following bioaccumulation of oral-dosed perfluorooctanoic acid: Loss of the Golgi apparatus

Despite its widespread use as a stabilizer across various industries over the past several decades, the health effects of chronic exposure to PFOA are still unclear. We administered PFOA by oral gavage (0, 12.5, 50, and 200 μg/day/mouse, eight groups) to male and female mice for six months. Body weight gain decreased with dose accompanied by increased liver weight, and PFOA altered liver damage-related-blood biochemical indicators and induced pathological lesions, including hepatocellular hypertrophy, cholangiofibrosis, and centrilobular hepatocellular vacuolation. Loss of the Golgi apparatus, formation of lamellar body-like structures, and lipid accumulation were observed in the liver of PFOA-treated mice. We also cohabited five pairs of male and female mice for the last ten days of administration, dosed PFOA to dam up to 28 days after birth, and investigated effects on reproduction and development. The survival rate of pups and the sex ratio of surviving mice decreased significantly at the highest dose. PFOA tissue concentration increased with the dose in the parent mice's liver and the pups' blood and brain. Taken together, we suggest that PFOA primarily affects the liver and reproduction system and that disturbance in lipid metabolism and Golgi's structural stability may be involved in PFOA-induced toxicity.

Evaluating the mode of action of perfluorooctanoic acid-induced liver tumors in male Sprague-Dawley rats using a toxicogenomic approach

The mode of action (MOA) underlying perfluorooctanoic acid (PFOA)-induced liver tumors in rats is proposed to involve peroxisome proliferator-activated receptor α (PPARα) agonism. Despite clear PPARα activation evidence in rodent livers, the mechanisms driving cell growth remain elusive. Herein, we used dose-responsive apical endpoints and transcriptomic data to examine the proposed MOA. Male Sprague-Dawley rats were treated with 0, 1, 5, and 15 mg/kg PFOA for 7, 14, and 28 days via oral gavage. We showed PFOA induced hepatomegaly along with hepatocellular hypertrophy in rats. PPARα was activated in a dose-dependent manner. Toxicogenomic analysis revealed six early biomarkers (Cyp4a1, Nr1d1, Acot1, Acot2, Ehhadh, and Vnn1) in response to PPARα activation. A transient rise in hepatocellular DNA synthesis was demonstrated while Ki-67 labeling index showed no change. Transcriptomic analysis indicated no significant enrichment in pathways related to DNA synthesis, apoptosis, or the cell cycle. Key cyclins including Ccnd1, Ccnb1, Ccna2, and Ccne2 were dose-dependently suppressed by PFOA. Oxidative stress and the nuclear factor-κB signaling pathway were unaffected. Overall, evidence for PFOA-induced hepatocellular proliferation was transient within the studied timeframe. Our findings underscore the importance of considering inter-species differences and chemical-specific effects when evaluating the carcinogenic risk of PFOA in humans.

A 13-week subchronic toxicity study of linalool oxide in Crl:CD(SD) rats.

Linalool oxide is frequently used as a flavoring agent, however, data on its toxicity is limited. In this study, we performed a 13-week subchronic toxicity study of linalool oxide (furanoid) in male and female Crl:CD(SD) rats. Doses of 0, 80, 250, and 800 mg/kg body weight (bw) per day were orally administered by gavage, using corn oil as the vehicle. Abnormal gait in both sexes and decreased locomotor activity in males were observed in the 800 mg/kg group. Reduced body weight gain was noted in both sexes at 800 mg/kg and at 250 mg/kg in males. In the 800 mg/kg group, serum biochemistry showed increased γ-glutamyl transpeptidase and decreased glucose in both sexes, increased total protein in males, and increased total cholesterol and phospholipids in females, suggesting that linalool oxide may have adverse effects on the liver. Increased relative and/or absolute liver weights, centrilobular hepatocellular hypertrophy in both sexes, and periportal microvesicular fatty changes in females were observed in the 800 mg/kg group. Increased relative liver weights and decreased serum glucose levels were observed in the 250 mg/kg male and female groups, respectively. Increased serum magnesium levels and relative kidney weights were observed in both sexes in the 800 mg/kg group, suggesting possible adverse effects of linalool oxide. Although histopathology showed accumulation of hyaline droplets in the male kidneys, immunohistochemistry revealed α2u-globulin nephropathy, which was not considered toxicologically significant. These results indicate that the no-observed-adverse-effect level of linalool oxide was 80 mg/kg bw/day for both sexes.

Safety of Elixinol Hemp Extract: In Vitro Genetic Toxicity and Subchronic Toxicity in Rats.

The results of safety studies performed with Elixinol Hemp Extract, a blend of hemp extract, cannabidiol (CBD) isolate, and copaiba containing approximately 65% total CBD, are described in this paper. In a 15-day range-finding study in rats, there were no effects of treatment with up to 101.4 mg/kg bw/day of the extract by gavage on any safety parameter measured in the study, with the exception that centrilobular hepatocellular hypertrophy occurred in all treatment groups, which correlated with increases in absolute liver weight in high-dose females and liver to terminal body weight ratio in mid-dose and high-dose females. A GLP-compliant 90-day OECD Guideline 408 study in rats that included a behavioral battery and a 28-day recovery phase was also conducted with Elixinol Hemp Extract administered by gavage. The doses used in the 90-day study were 0 (vehicle), 28.94, 50.64, and 86.81 mg/kg bw/day. The findings were similar to those observed in the range-finding study. There were no effects of the test material on any test parameter in the 90-day study other than findings related to the liver (increased liver weight in high-dose main study males and mid-dose and high-dose main study females and low incidences of hepatocellular hypertrophy and vacuolation in main study high-dose males). Similar findings were not observed in the recovery animals, and there were no alterations in the clinical chemistry suggestive of liver toxicity in any of the main study or recovery animals. Therefore, the liver outcomes observed in the main study were not considered adverse. The test material also tested negative for mutagenicity in bacterial reverse mutation assays (plate incorporation and preincubation) in the absence and presence of metabolic activation. The results indicate that the oral 90-day no observed adverse effect level (NOAEL) of Elixinol Hemp Extract in rats is 86.81 mg/kg bw/day (highest dose administered), and that the extract is not mutagenic.

Evaluation of the Effect of Perfluorohexane Sulfonate on the Proliferation of Human Liver Cells.

Perfluorohexane sulfonate (PFHxS) is a widely detected replacement for legacy long-chain perfluoroalkyl substances (PFAS) in the environment and human blood samples. Its potential toxicity led to its recent classification as a globally regulated persistent organic pollutant. Although animal studies have shown a positive association between PFHxS levels and hepatic steatosis and hepatocellular hypertrophy, the link with liver toxicity, including end-stage liver cancer, remains inconclusive. In this study, we examined the effects of PFHxS on the proliferation of Hep3B (human hepatocellular carcinoma) and SK-Hep1 (human liver sinusoidal endothelial cells). Cells were exposed to different PFHxS concentrations for 24-48 h to assess viability and 12-14 days to measure colony formation. The viability of both cell lines increased at PFHxS concentrations <200 μM, decreased at >400 μM, and was highest at 50 μM. Colony formation increased at <300 μM and decreased at 500 μM PFHxS. Consistent with the effect on cell proliferation, PFHxS increased the expression of proliferating cell nuclear antigen (PCNA) and cell-cycle molecules (CDK2, CDK4, cyclin E, and cyclin D1). In summary, PFHxS exhibited a biphasic effect on liver cell proliferation, promoting survival and proliferation at lower concentrations and being cytotoxic at higher concentrations. This suggests that PFHxS, especially at lower concentrations, might be associated with HCC development and progression.

Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis.

The liver has multiple regeneration modes, including hepatocellular hypertrophy and self-renewal of hepatocytes. When hepatocyte proliferation is impaired, hepatic progenitor cells may proliferate through ductular reaction (DR), differentiate into hepatocytes, and contribute to fibrosis. However, the three-dimensional spatial relationship between DR and regenerating hepatocytes and dynamic changes in DR associated with fibrosis remain poorly understood. Here, we performed three-dimensional (3D) imaging of cleared 42 liver explants with chronic and acute liver diseases and 4 normal livers to visualize DR. In chronic hepatic liver diseases, such as viral hepatitis, steatohepatitis, autoimmune hepatitis, and cryptogenic cirrhosis, the total length and number of branches of DR showed a significant positive correlation. We studied the spatial relationship between DR and GS-expressing cells using glutamine synthetase (GS) and cytokeratin 19 (CK19) as markers of liver regeneration and DR, respectively. The percentage of CK19-positive cells that co-expressed GS was less than 10% in chronic liver diseases. In contrast, nearly one-third of CK19-positive cells co-expressed GS in acute liver diseases, and chronic cholestatic liver diseases, including primary biliary cholangitis and primary sclerosing cholangitis, showed no co-expression. We also found that DR was longer and had more branching in livers with progressive fibrosis compared to those with regressive fibrosis. Our results suggest that DR displays varying degrees of spatial complexity and contribution to liver regeneration. DR may serve as hepatobiliary junctions that maintain continuity between hepatocytes and bile ducts rather than hepatocyte regeneration in chronic liver diseases.

Pyridylnidulin exerts anti-diabetic properties and improves non-alcoholic fatty liver disease in diet-induced obesity mice.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is one of the metabolic disorders related to the pathophysiology of type 2 diabetes mellitus (T2DM). Therapeutic strategies are focused on the improvement of energy balance and lifestyle modification. Additionally, the derivative of the bioactive fungal metabolite is of interest to provide health benefits, especially in obese and pre-diabetic conditions. In our screening of anti-diabetic compounds from fungal metabolites and semisynthetic derivatives, a depsidone derivative, namely pyridylnidulin (PN), showed potent glucose uptake-inducing activity. The present study aimed to investigate the liver lipid metabolism and anti-diabetic properties of PN in diet-induced obesity mice. Methods: Male C57BL/6 mice were induced obesity and pre-diabetic conditions by dietary intervention with a high-fat diet (HFD) for 6 weeks. These obese mice were orally administered with PN (40 or 120 mg/kg), metformin (150 mg/kg), or vehicle for 4 weeks. Glucose tolerance, plasma adipocytokines, hepatic gene and protein expressions were assessed after treatment. Results: Improved glucose tolerance and reduced fasting blood glucose levels were found in the PN and metformin-treated mice. Additionally, hepatic triglyceride levels were consistent with the histopathological steatosis score regarding hepatocellular hypertrophy in the PN and metformin groups. The levels of plasma adipocytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were reduced in the PN (120 mg/kg) and metformin-treated mice. In addition, hepatic gene expression involved in lipid metabolism, including lipogenic enzymes was significantly reduced in the PN (120 mg/kg) and metformin-treated mice. The increased protein expression levels of phosphorylated AMP-activated protein kinase (p-AMPK) was also found in PN and metformin-treated mice. Discussion: Considering the increased p-AMPK protein expression levels in PN and metformin-treated mice were revealed as the underlying mechanisms to improve metabolic parameters. These results suggested that PN provided the health benefit to slow the progression of NAFLD and T2DM in obese and pre-diabetic conditions.

Effects of perfluorooctanoic acid (PFOA) on gene expression profiles via nuclear receptors in HepaRG cells: Comparative study with in vitro transactivation assays

Perfluorooctanoic acid (PFOA), a synthetic perfluorinated eight-carbon organic chemical, induces hepatotoxicity in rodents, indicated increased liver weight, hepatocellular hypertrophy, necrosis, and peroxisome proliferation. Epidemiological studies have demonstrated the association between serum PFOA levels and various adverse effects. In this study, we investigated the gene expression profiles of human HepaRG cells exposed to 10 and 100 μM PFOA for 24 h. Treatment with 10 and 100 μM PFOA significantly modulated the expression of 190 and 996 genes, respectively. Genes upregulated or downregulated by 100 µM PFOA included peroxisome proliferator-activated receptor (PPAR) signaling genes related to lipid metabolism, adipocyte differentiation, and gluconeogenesis. Moreover, we identified the “Nuclear receptors-meta pathways” following the activation of other nuclear receptors: constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR), as well as the transcription factor nuclear factor E2-related factor 2 (Nrf2). The expression levels of some target genes (CYP4A11, CYP2B6, CYP3A4, CYP7A1, and GPX2) of these nuclear receptors and Nrf2 were confirmed using quantitative reverse transcription polymerase chain reaction. Next, we performed transactivation assays using COS-7 and HEK293 cells to investigate whether these signaling-pathways were activated by the direct effects of PFOA on human PPARα, CAR, PXR, FXR and Nrf2. PFOA concentration-dependently activated PPARα, but not CAR, PXR, FXR, or Nrf2. Taken together, these results suggest that PFOA affects the hepatic transcriptomic responses of HepaRG cells through the direct activation of PPARα and indirect activation of CAR, PXR, FXR, and Nrf2. Our finding indicates that PPARα activation in the “Nuclear receptors-meta pathways” functions as a molecular initiating event for PFOA, and indirect activation of alternative nuclear receptors and Nrf2 also induce important molecular mechanisms in PFOA-induced human hepatotoxicity.

Reproductive and developmental toxicity evaluation of cannabidiol.

An important data gap in determining a safe level of cannabidiol (CBD) intake for consumer use is determination of CBD's potential to cause reproductive or developmental toxicity. We conducted an OECD Test Guideline 421 GLP-compliant study in rats, with extended postnatal dosing and hormone analysis, where hemp-derived CBD isolate (0, 30, 100, or 300 mg/kg-bw/d) was administered orally. Treatment-related mortality, moribundity, and decreased body weight and food consumption were observed in high-dose F0 adult animals, consistent with severe maternal toxicity. No effects were observed on testosterone concentrations, F0 reproductive performance, or reproductive organs. Hepatocellular hypertrophy in the 100- and 300 mg/kg-bw/day groups correlated with hypertrophy/hyperplasia in the thyroid gland and changes in mean thyroid hormone concentrations in F0 animals. Mean gestation length was unaffected; however, total litter loss for two females and dystocia for two additional females in the high-dose group occurred. Other developmental effects were limited to lower mean pup weights in the 300 mg/kg-bw/d group than those of controls. The following NOAELs were identified for CBD isolate based on this study: 100 mg/kg-bw/d for F0 systemic toxicity and female reproductive toxicity, 300 mg/kg-bw/d for F0 male reproductive toxicity, and 100 mg/kg-bw/d for F1 neonatal and F1 generation toxicity.

Comprehensive analysis of the general toxicity, genotoxicity, and carcinogenicity of 3-acetyl-2,5-dimethylfuran in male gpt delta rats.

The safety of flavoring agents has been evaluated according to classification by chemical structure and using a decision tree approach. The genotoxic potential found in some flavoring agents has highlighted the importance of efficient toxicity studies. We performed a comprehensive toxicity analysis using reporter gene transgenic rats to assess the safety of 3-acetyl-2,5-dimethylfuran (ADF), a flavoring agent exhibiting genotoxic potential in silico and in vitro assays. Male F344 gpt delta rats were given 0, 30, or 300mg/kg body weight/day ADF by gavage for 13 weeks. In serum biochemistry analyses, triglyceride, total cholesterol, phospholipid, and total protein levels and albumin/globulin ratios were significantly altered in the 30 and 300mg/kg groups. Histopathologically, nasal cavity toxicity and hepatocellular hypertrophy were observed in the 300mg/kg group. In the livers of 300mg/kg group, a significant increase in gpt mutant frequencies were observed along with ADF-specific DNA adduct formation. The number and area of glutathione S-transferase placental form-positive foci were significantly increased in the same group. Thus, ADF affected nasal cavity, liver, and lipid metabolism and showed genotoxicity and possible carcinogenicity in the liver. Overall, our comprehensive toxicity study using gpt delta rats provided insights into the safety evaluation of ADF.

Gene expression analysis of antioxidant and DNA methylation on the rat liver after 4-week wood preservative chromated copper arsenate exposure.

Our previous 4-week repeated dose toxicity study showed that wood preservative chromated copper arsenate (CCA) induced hepatocellular hypertrophy accompanied by biochemical hepatic dysfunction and an increase in oxidative stress marker, 8-hydroxydeoxyguanosine, in female rats. To further explore the molecular mechanisms of CCA hepatotoxicity, we analyzed 10%-buffered formalin-fixed liver samples from female rats for cell proliferation, apoptosis, and protein glutathionylation and conducted microarray analysis on frozen liver samples from female rats treated with 0 or 80 mg/kg/day of CCA. Chemical analysis revealed that dimethylated arsenical was the major metabolite in liver tissues of male and female rats. CCA increase labeling indices of proliferating cell nuclear antigen and decrease terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling accompanied with increased expression of protein glutathionylation, indicating a decrease in glutathione (GSH) in hepatocytes of female rats. Microarray analysis revealed that CCA altered gene expression of antioxidants, glutathione-S-transferase (GST), heat shock proteins and ubiquitin-proteasome pathway, cell proliferation, apoptosis, DNA methylation, cytochrome P450, and glucose and lipid metabolism in female rats. Increased expression of GSTs, including Gsta2, Gsta3, Mgst1, and Cdkn1b (p27), and decreased expression of the antioxidant Mt1, and DNA methylation Dnmt1, Dnmt3a, and Ctcf were confirmed in the liver of female rats in a dose-dependent manner. Methylation status of the promoter region of the Mt1 was not evidently changed between control and treatment groups. The results suggested that CCA decreased GSH and altered the expression of several genes, including antioxidants, GST, and DNA methylation, followed by impaired cell proliferation in the liver of female rats.

Therapeutic Effects of Heterotrigona itama (Stingless Bee) Bee Bread in Improving Hepatic Lipid Metabolism through theActivation of the Keap1/Nrf2 Signaling Pathway in an Obese Rat Model.

Bee bread (BB) has traditionally been used as a dietary supplement to treat liver problems. This study evaluated the therapeutic effects of Heterotrigona itama BB from Malaysia on obesity-induced hepatic lipid metabolism disorder via the regulation of the Keap1/Nrf2 pathway. Male Sprague Dawley rats were fed with either a normal diet or high-fat diet (HFD) for 6 weeks to induce obesity. Following 6 weeks, obese rats were treated either with distilled water (OB group), BB (0.5 g/kg body weight/day) (OB + BB group) or orlistat (10 mg/kg body weight/day) (OB + OR group) concurrent with HFD for another 6 weeks. BB treatment suppressed Keap1 and promoted Nrf2 cytoplasmic and nuclear translocations, leading to a reduction in oxidative stress, and promoted antioxidant enzyme activities in the liver. Furthermore, BB down-regulated lipid synthesis and its regulator levels (SIRT1, AMPK), and up-regulated fatty acid β-oxidation in the liver of obese rats, being consistent with alleviated lipid levels, improved hepatic histopathological changes (steatosis, hepatocellular hypertrophy, inflammation and glycogen expression) and prevented progression to non-alcoholic steatohepatitis. These results showed the therapeutic potentials of H. itama BB against oxidative stress and improved lipid metabolism in the liver of obese rats possibly by targeting the Keap1/Nrf2 pathway, hence proposing its role as a natural supplement capable of treating obesity-induced fatty liver disease.

Acute and Subchronic Oral Toxicity of Anthraquinone in Sprague Dawley Rats.

Objective: This study was conducted to evaluate the acute and subchronic toxicity of anthraquinone. An acute toxicity test was performed in female Sprague Dawley (SD) rats, and the oral median lethal dose (LD50) of anthraquinone was estimated to be >5000 mg/kg body weight (BW). In the subchronic study, groups of 10 male and 10 female rats were dosed with anthraquinone by gavage at 0, 1.36, 5.44, 21.76, and 174.08 mg/kg BW, 7 days/week for 90 days followed by a recovery period of 28 days. No appreciable toxic-related changes were observed in the 1.36 mg/kg BW group. When the animals received 5.44 mg/kg BW or more of anthraquinone, hyaline droplet accumulation in the renal tubules was observed in both the male and female rats, and anemia was observed in the females. When the anthraquinone dose reached 174.08 mg/kg BW, mild hepatocellular hypertrophy around the central vein of the hepatic lobule and hypothyroidism were observed in the female rats. During the recovery period, changes in clinical symptoms and parameters were considerably alleviated. Based on the results of this study, the no observed adverse effect level (NOAEL) for anthraquinone in rats was set at 1.36 mg/kg BW, and the lowest observed adverse effect level (LOAEL) was 5.44 mg/kg BW.

Seventy-Day Toxicity Study in Juvenile Sprague-Dawley Rats with Semicarbazide (SEM) from Weaning to Sexual Maturity.

The study was conducted to evaluate the toxicological effects, functional observation battery tests, and sexual maturity of semicarbazide oral gavage administration to juvenile Sprague-Dawley rats for 70 days at 0, 15, 30, and 60 mg/kg/day weaning to sexual maturity. At 60 mg/kg/day, there was a delay in mean age at acquisition of balano-preputial and vaginal patency and a decrease in body weight and food consumption in males. Treatment increased reticulocyte count, aspartate aminotransferase, and alanine aminotransferase levels in both sexes and decreased hematocrit and protein in males. Increased absolute and relative liver and spleen weight in both sexes were observed. Male rats had lower thymus and testes weights, whereas female rats had lower uterine weights. Semicarbazide caused significant changes in sperm motility, sperm count, and sperm abnormality. Histopathologically, semicarbazide caused cortical hypertrophy in adrenals and increased extramedullary hematopoiesis in the spleen; hepatocellular hypertrophy, follicular epithelial hypertrophy in the thyroid, and degeneration of seminiferous tubules in the testis were observed at 60 mg/kg/day when compared to control. Results suggest that 60 mg/kg/day of semicarbazide can exert systemic toxicity in juvenile rats. The no observed adverse effect level (NOAEL) of semicarbazide for juvenile Sprague-Dawley rats was estimated to be 30 mg/kg/day.

Risk Assessment of the Pesticide Coragen 20 SC with the Active Substance Chlorantraniliprole

Coragen 20 SC is a new product in Norway containing the active substance chlorantraniliprole. It is applied for use in apples against codling moth (Cydia pomonella), apple fruit moth (Argyresthia conjugella) and free leaf living larvae. The Norwegian Scientific Committee for Food Safety (VKM) was asked by the Norwegian Food Safety Authority to perform a risk assessment on human health, environmental fate and ecotoxicological of the active substance and the product. The risk assessment of the product was approved at a meeting 11th of May 2010 by VKMs Scientific Panel on Pesticides (Panel 2). VKMs Panel 2 concludes as following: The product and the active substance have low acute oral, dermal and inhalation toxicities. Both are non-irritating to the skin, and no allergenic potential by skin contact were shown. Coragen was non-irritating to the eyes, while chlorantraniliprole showed a weak irritating potential. Chlorantraniliprole is not shown to have any genotoxic or carcinogenic potential, or to be teratogenic or toxic to the reproduction of female animals. The potential for testicular toxicity of chlorantraniliprole is unclear because the study design and the limited number of young dogs (2/sex/group) do not provide basis for a firm conclusion. No particular target organ for toxicity in any species in the sub-chronic and chronic toxicity studies was seen. The observed dose- and time dependent increased degree of microvesiculation in the zona fasiculata of the adrenal cortex in male rats, is however of uncertain biological significance. All test species (rat, mice, dog) showed physiological adaption to chlorantraniliprole administration (increased liver metabolism with induction of cytochrome P450 enzymes) which was manifested as increased liver weight and hepatocellular hypertrophy. In the chronic toxicity study in mice, the increased liver weight and hepatocellular hypertophy was accompanied with eosinofilic foci, which was assessed as an adverse effect. The no observed effect level (NOAEL) derived from this study serves as basis for calculations of values for acceptable daily intake (ADI) and acceptable operator exposure level (AOEL). &#x0D; In Panel 2‘s opinion a sub-chronic study (90 days) with the technical material (E2Y45-282) including relevant concentration of the impurity IN-G2S78 should be performed. This would bring information on possible influence of the impurity on the toxicological profile of the technical material, and consequently on the assessment of the NOAELs in the various toxic studies. &#x0D; The estimated risk for operator and for bystanders or for workers re-entering treated crops is assessed as minimal. &#x0D; Chlorantraniliprole is persistent in soil with half live of about 1 year. The long half life indicates high potential for accumulation in soil after repeated use, which is confirmed by both model simulations and field studies. The Panel considers field data from the south of Europe not to be relevant for the Nordic conditions based on different climate conditions and soil properties contributing in different directions.&#x0D; The Panel considers that the existing documentation is not sufficient for a firm conclusion on the use of normalised field data for modelling purposes. However, the substance is persistent and expected to accumulate in soil. The Panel concludes that there is minimal risk for toxic effects on mammals, birds, bees, and microorganisms in soil due to chlorantraniliprole exposure with the proposed exposure regime. Panel concludes that there is “very high risk” for effects on in-field non target arthropods from chlorantraniliprole exposure. &#x0D; For soil living invertebrates, earthworms seem rather insensitive to chlorantraniliprole and the Panel considers the toxic effects to be minimal. Panel considers the risk for toxic effects on soil living arthropods to be high. Crustaceans and insects larvae are the aquatic organisms most sensitive to chlorantraniliprole. The Panel concludes that there is a minimal risk of toxic effects on aquatic organisms due to exposure to chlorantraniliprole with the proposed application regime provided that a buffer zone of 30 m to surface water is applied.

Neonatal Oral Administration of Chrysin Prevents Long-Term Development of Non-Alcoholic Fatty Liver Disease in a Sexually Dimorphic Manner in Fructose Nurtured Sprague Dawley Rats.

High-fructose diets are linked with the development of non-alcoholic fatty liver disease (NAFLD), the management of which is a burden to society. Interventions with phytochemicals in the early postnatal period may prevent fructose-induced NAFLD later in adulthood. We investigated the protective potential of chrysin against fructose-induced NAFLD. Four-day-old male and female suckling Sprague Dawley rats (N = 112) were randomly grouped and orally gavaged daily with distilled water (negative Control-Cn + W), chrysin(Chr-100 mg/kg), fructose-solution (Fr-20% w/v), and Chr + Fr between postnatal day (PND) 4 and 21 and then weaned onto normal rat chow and plain drinking water to PND 55. From PND 56 to 130, half of the rats continued on plain water, and the rest had Fr as drinking fluid. Terminally, the liver tissue was collected, and the lipid content was determined and histologically assessed for NAFLD. Dietary Fr induced an increased hepatic lipid content (p = 0.0001 vs. Cn + W) both sexes, and it was only attenuated by neonatal Chr in female rats (p < 0.05). Histologically, there was increased microvesicular steatosis (p = 0.0001 vs. Cn + W) in both sexes, and it was prevented by neonatal Chr (p > 0.05). Fr caused macrovesicular steatosis (p = 0.01 vs. Cn + W) in females only, and chrysin did not prevent it (p > 0.05). Fr induced hepatocellular hypertrophy, and inflammation was observed in females only (p = 0.01 vs. Cn + W), and this was prevented by Chr (p > 0.05). The collagen area fraction was increased by Fr (p = 0.02 (males) and p = 0.04 (females) vs. Cn + W, respectively; however, chrysin did not prevent this (p > 0.05). Neonatal chrysin prevented some of the deleterious effects of the high-fructose diet on the liver, suggesting that chrysin should be further explored as a strategic prophylactic neonatal intervention against high-fructose-diet-induced NAFLD.

The sub-acute toxicity of kavalactone in rats: a study of the effect of oral doses and the mechanism of toxicity in combination with ethanol

Kava is a herbal supplement and beverage made from the Piper methysticum plant, which is known for its recreational use as a mood enhancer, relaxation, as well as pain relief for centuries. Kava is widely used among alcoholics, but it is dangerous and potentially fatal. The objectives of this study were to examine the sub-acute toxicity effects of different doses of 70% kavalactone (KL) in rats by oral application, as well as to elucidate the mechanisms of toxicity alone and in combination with ethanol (EtOH). The most common side effects observed were abnormal breathing, ataxia, lethargy, loss of appetite, indigestion, and loss of coordination, especially in the 800 mg/kg bw, po bodyweight dosage of kava treatment group alone, and in combination with EtOH. In the sub-acute study, there were dose-related decreases in body weight, feed intake, and water consumption rates. Gross and histopathological findings revealed that the liver was abnormal in color, size, consistency, and the weight significantly increased at a dose of 800 mg/kg bw, po, with KL alone and a greater increase in combination with EtOH. Hepatocellular hypertrophy (HP) and necrosis with Kupffer cells hyperplasia were observed in the periacinar zone of all rats dosed with KL (800 mg/kg bw, po) alone, and extensive changes were observed in combination with EtOH. The periportal (Z1) and mid-zonal (Z2) areas of hepatocytes were less affected as compared to the periacinar zone. These results demonstrate that EtOH exacerbated the sedative and hypnotic activity of KL, and markedly increased toxicity. The histopathological results supported the clinical and biochemical findings and the severity of hepatic damage in a dose-dependent manner.

Limosilactobacillus fermentum MG4295 Improves Hyperglycemia in High-Fat Diet-Induced Mice.

Hyperglycemia due to uncontrolled glucose regulation is widely known as cause of diabetes, non-alcoholic fatty liver disease (NAFLD), and other complications. NAFLD refers to a condition in which fat is excessively accumulated, whether inflamed or not, and has caused serious medical problems in recent years. The aim of this study was to explore the antihyperglycemia effects of Limosilactobacillus fermentum MG4295 (L. fermentum MG4295) in high-fat diet (HFD)-induced in vivo. We demonstrated the suitability of L. fermentum MG4295 as a probiotic by observing its stability, survivability, and proliferation under simulated gastrointestinal conditions, and safety, antibiotic susceptibility, hemolysis, and enzyme activity. The potential antihyperglycemic activity of L. fermentum MG4295 was investigated in an HFD and sugar-water-induced mouse model. Administration of this strain for 12 weeks showed an improved trend in glucose tolerance, insulin, alanine amino transferase, total cholesterol, low-density lipoprotein cholesterol, and glucagon-like peptide-1. Histopathological analysis revealed that L. fermentum MG4295 significantly reduced the histopathological scores of hepatic steatosis, inflammation, and hepatocellular hypertrophy in liver tissues and lipid content in adipose tissues. Administration of L. fermentum MG4295 upregulated IRS-1, AKT, and GLUT4 and downregulated G6Pc and PEPCK expression in liver and/or muscle tissues. Our results suggest that L. fermentum MG4295 can improve hyperglycemia. Furthermore, it can be used as a dietary functional supplement to manage blood glucose.

Toxicity and toxicokinetic assessment of an anti-tubercular drug pretomanid in cynomolgus monkeys

Pretomanid is a nitroimidazooxazine antimycobacterial drug that was approved in more than 10 countries as part of a three-drug, all oral regimen, consisting of bedaquiline, pretomanid, and linezolid (BPaL) for 6-months treatment of adults with pulmonary extensively drug-resistant tuberculosis (XDR-TB) or with complicated forms of multidrug-resistant tuberculosis (MDR-TB). The toxicological profile of pretomanid was thoroughly evaluated in repeat-dose oral toxicity studies up to 39 weeks long in cynomolgus monkeys. Exposures up to 10-fold higher than in humans at the approved pretomanid dose (200 mg) were achieved in acute studies allowing for characterization of dose-limiting toxicity. Target organs and processes identified in acute and chronic toxicity studies included QT prolongation, nervous system effects, and liver effects (minimal hepatocellular hypertrophy without elevations in liver enzymes). In a 13-week study, no cataracts were present at the end of dosing, but 2 of 12 monkeys had cataracts at the end of a 13-week recovery period. No cataracts related to pretomanid administration were observed in subsequent 13-week or 39-week studies. No male reproductive toxicity was observed in these studies. No-observed-adverse-effect levels (NOAELs) were identified in all studies. Exposures at the NOAELs equaled, or exceeded, human exposure at the approved pretomanid dose with the exception of female monkeys in a 39-week chronic toxicity study. These data support the use of pretomanid as part of the 6-month BPaL regimen for treating XDR-TB and MDR-TB.