30-day Recovery Period Research Articles

Cytokinin and gibberellic acid-mediated waterlogging tolerance of mungbean (Vigna radiata L. Wilczek).

BackgroundMungbean (Vigna radiata L. Wilczek) is one of the most important pulse crops, well-known for its protein-rich seeds. Growth and productivity are severely undermined by waterlogging.MethodsIn this study, we aim to evaluate how two promising phytohormones, namely cytokinin (CK) and gibberellic acid (GA3), can improve waterlogging tolerance in mungbean by investigating key morphological, physiological, biochemical, and yield-related attributes.ResultsOur results showed that foliar application of CK and GA3 under 5-day of waterlogged conditions improved mungbean growth and biomass, which was associated with increased levels of photosynthetic rate and pigments. Waterlogged-induced accumulation of reactive oxygen species and the consequently elevated levels of malondialdehyde were considerably reduced by CK and GA3 treatments. Mungbean plants sprayed with either CK or GA3 suffered less oxidative stress due to the enhancement of total phenolics and flavonoids levels. Improvement in the contents of proline and total soluble sugars indicated a better osmotic adjustment following CK and GA3 treatments in waterlogged‐exposed plants. Most fundamentally, CK or GA3-sprayed waterlogged-stressed mungbean plants demonstrated better performance in the aforementioned parameters after the 15-day recovery period as compared to water-sprayed waterlogged-exposed plants. Our results also revealed that CK and GA3 treatments increased yield-associated features in the waterlogged-stressed plant. Here, both phytohormones are efficient in improving mungbean resistance to waterlogging. However, CK was found to be more effective. Overall, our findings suggested that CK or GA3 could be used for managing waterlogging-induced damage to mungbean and perhaps in other cash crops.

Ninety-day repeated oral toxicity study of saponified Capsicum annum fruit extract with 50% capsanthin in Sprague-Dawley Rats with a 28-day recovery period

sA ninety-day oral toxicity study of saponified Capsicum annum fruit extract with 50% (w/w) capsanthin (SCFE-50 C) was performed by oral gavage administration to male and female Sprague-Dawley (SD) rats at doses of 0, 500, 1000 and 2000 mg/kg BW/day for a period of ninety consecutive days. To assess the reversal of toxicity, the treatment phase was followed with a twenty-eight-day recovery period. The treatment with SCFE-50 C in both male and female SD rats showed no mortality, and no treatment-related toxicologically significant changes were observed in any groups. No significant differences between treated and control groups were found in feed consumption, body weight gain, individual organ weights, ocular examination, clinical chemistry or blood biochemistry. The necroscopy and histopathology examination did not reveal any clinically significant changes in male and female rats from the 2000 mg/kg BW/day group. According to this study, the no observable adverse effect level (NOAEL) for saponified Capsicum annum fruit extract with 50% (w/w) capsanthin (SCFE-50 C) administered by oral gavage for 90-days is > 2000 mg/kg BW/day in SD rats.

B-AB03-02 BIPHASIC FOCAL MONOPOLAR PULSED ELECTRIC FIELD THERAPY: PRESERVED PHRENIC NERVE FUNCTION AND ANATOMY

Thermal cardiac ablation may cause phrenic nerve (PhN) palsy. Pulsed electric fields (PEF) induce cell death independently from thermal processes, potentially enabling cardiac ablation underlying the PhN without disrupting its function or morphology. This preclinical study evaluates cardiac-based PEF treatments on the morphology and function of the PhN. In the right atrium of 4 pigs a focal ablation catheter was maneuvered with pacing until the location and minimum PhN stimulation threshold was established, as determined by monitoring diaphragmatic movement while decreasing pacing amplitude until capture was lost. Biphasic monopolar PEF therapy was then delivered with the focal catheter either directly over, or in proximity to the PhN when electrically isolating the SVC and RSPV. Following treatment, the PhN stimulation threshold was re-evaluated. Pigs were survived acutely (n=1) and at 28 days (n=3), after which the PhN was examined grossly and histologically. The PhN stimulation threshold did not change following treatment. No clinical signs of PhN palsy nor diaphragmatic paralysis were observed during the 28-day recovery period. Gross and histological examination of the PhN did not indicate signs of macroscopic nor microscopic morphology, while SVC and RSPV electrical isolation was retained. The mechanisms underlying PEF ablation enable treatment to tissue adjacent to the PhN without adversely affecting its form or function. This suggests the ability to perform PEF-based cardiac ablation in regions typically associated with high risk for PhN injury without appreciable risk of PhN-associated morbidity.

Экспериментальное обоснование предельно допустимой концентрации дихлоргексафторбутена в воздухе рабочей зоны

To date, there have been no exposure standards for air concentrations of 1,4-dichlorohexafluorobutene (DCHF) in the work areas. The study was aimed to assess the toxicity of DCHF and to evaluate health hazard in acute, subacute, and chronic experiments. It was found that the substance was highly hazardous, DL50 in mice after intragastric injection was 79.0 mg/kg, СL50 was 229.0 mg/m3, and in rats these values were 86,0 mg/kg and 670,0 mg/m3. In animals, DCHF had a moderate local irritative effect on animal skin and ocular mucous membranes, as well as the skin resorptive effect. The 18.2 mg/m3 threshold limit concentration for a single inhalation exposure to DCHF was defined based on the changes in behavior responses and blood parameters. The 30-day subacute inhalation experiment revealed the pronounced cumulative effect of the substance. The 4-months chronic inhalation study showed that the exposure of experimental rats to 16.8 mg/m3 concentration of DCHF resulted in impaired function of central nervous system and cardiac activity, altered hematologic, biochemical, acid-base, and blood gas values, as well as in morphological alterations in lungs, which persisted after the 30-day recovery period. The chronic exposure threshold defined for DCHF was 2.2 mg/m3, and the defined no observable effect level was 0.24 mg/m3. Based on the study results, the maximum permissible concentration of DCHF in the air of the working area of 0.2 mg/m3 was confirmed and approved, the substance was assigned hazard class 2, vapor + aerosol + (specific protection of skin and eyes required). Gas chromatographic method using electron-capture detection for determination of DCHF mass air concentration in the work areas has been developed and approved.

Biological effects of multimetal (Ni, Cd, Pb, Cu, Cr, Zn) mixture in rainbow trout Oncorhynchus mykiss: Laboratory exposure and recovery study

The present study tested the biological consequences of exposure to a multimetal mixture as a multiple chemical stressor on Oncorhynchus mykiss at molecular, cellular, physiological and whole-organism levels and on biomarker responses of this fish during the depuration period. To represent environmentally relevant multiple chemical stressors, in our study, we used the mixture of Zn, Cu, Ni, Cr, Pb and Cd at the concentrations corresponding to Maximum-Permissible-Concentrations (MPCs) acceptable for the EU inland waters. This study was undertaken with a view to elucidate if changes in the MPC of the test mixture components (Ni, Pb, Cd) could cause significantly different biomarker responses in O. mykiss from those previously determined in the carnivorous and omnivorous fishes exposed to the mixture of the same metals but at different MPCs of Ni, Pb and Cd. This study has revealed that exposure to mixtures of metals at MPC produces genotoxic effects in fish blood erythrocytes and a lethargic effect on O. mykiss behaviour, and, also, significantly increases the levels of Cd, Cr and Ni accumulated in the gills tissue. O. mykiss successfully depurated Cr and Ni in less than 28 days, however, the level of Cd decreased by only approximately 40% over the same period. A significant capacity of O. mykiss to restore its DNA integrity (Comet assay) after exposure to metal mixtures was revealed. However, the 28-day recovery period proved to be insufficiently long for erythrocytes with nuclear abnormalities to recover to the unexposed level. In conclusion, changes in the MPCs of Ni, Pb and Cd in the test mixture produce biological effects similar to those previously determined in S. salar, R. rutilus and P. fluviatilis exposed to the mixture of the same metals but at lower MPCs of Ni and Pb and at higher MPC of Cd.

Suppression of systemic inflammation and signs of acute and chronic cholangitis by multi-kinase inhibitor 1-(4-Cl-benzyl)-3-chloro-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione.

An aberrant activity of growth factor receptors followed by excessive cell proliferation plays a significant role in pathogenesis of cholangitis. Therefore, inhibition of these processes could be a fruitful therapeutic strategy. The effects of multi-kinase inhibitor 1-(4-Cl-benzyl)-3-chloro-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione (MI-1) on the hepatic and systemic manifestations of acute and chronic cholangitis in rats were addressed. MI-1 (2.7mg/kg per day) was applied to male rats that experienced α-naphthylisothiocyanate-induced acute (3days) or chronic (28days) cholangitis. Liver autopsy samples, blood serum markers, and leukograms were studied. MI-1 localization in liver cells and its impact on viability of HepG2 (human hepatoma), HL60 (human leukemia), and NIH3T3 (normal murine fibroblasts) cell lines and lymphocytes of human peripheral blood (MTT, DNA fragmentation, DNA comet assays, Propidium Iodide staining) were assessed. Under both acute and chronic cholangitis, MI-1 substantially reduced liver injury, fibrosis, and inflammatory scores (by 46-86%) and normalized blood serum markers and leukograms. Moreover, these effects were preserved after a 28-day recovery period (without any treatment). MI-1 inhibited the HL60, HepG2 cells, and human lymphocytes viability (IC50 0.6, 9.5 and 8.3µg/ml, respectively), while NIH3T3 cells were resistant to that. Additionally, HepG2 cells and lymphocytes being incubated with MI-1 demonstrated insignificant pro-apoptotic and pro-necrotic changes and DNA single-strand breaks, suggesting that MI-1 effects in liver might be partly caused by its cytotoxic action towards liver cells and lymphocytes. In conclusion, MI-1 attenuated the systemic inflammation and signs of acute and chronic cholangitis partly through cytotoxicity towards cells of hepatic and leukocytic origin.

Toxicological evaluation of the ketogenic ester bis hexanoyl (R)-1,3-butanediol: Subchronic toxicity in Sprague Dawley rats

Bis-hexanoyl (R)-1,3-butanediol (BH-BD) is novel ketone ester undergoing development as a food ingredient to achieve nutritional ketosis in humans. Male and female Crl:CD(SD) rats were administered BH-BD twice daily at 9000, 12,000 or 15,000 mg/kg/day, by oral gavage in a 90-day toxicity study with 28-day recovery period; and an interim 28-day phase. Test substance-related early deaths occurred in four females at 15,000 mg/kg/day. A dose-dependent increase in acute transient postdose (1–3 h) observations of incoordination at ≥12,000 mg/kg/day and decreased activity at all dose levels were noted in both sexes. Postdose observations were likely associated with peak ketonemia and were considered adverse at 15,000 mg/kg/day. These daily observations decreased over the study without any persistent effects, as determined during weekly pre-dose observations. Adverse histopathological changes included ulceration/erosion in non-glandular stomach at ≥ 12,000 mg/k/day and in glandular stomach at 15,000 mg/kg/day. These histopathological findings were not noted after 28-days of recovery. Due to unlikely human relevance of the rat non-glandular stomach effects for BH-BD and test substance-related mortality at 15,000 mg/kg/day, the no-observed-adverse-effect level (NOAEL) for subchronic toxicity of BH-BD was determined to be 12,000 mg/kg/day.

Safety evaluation after acute and sub-chronic oral administration of high purity nicotinamide mononucleotide (NMN-C®) in Sprague-Dawley rats

β-nicotinamide mononucleotide (NMN) is a natural molecule intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Preclinical evidences point to the beneficial effect of NMN administration on several age-related conditions. The present work aimed at studying mutagenicity, and genotoxicity, acute oral toxicity and subchronic oral toxicity of a high purity synthetic form of NMN (NMN-C®) following the OECD guidelines. In the experimental conditions tested, NMN-C® was not mutagenic or genotoxic. Acute toxicity assay revealed that at an oral limit dose of 2666 mg/kg, NMN-C® did not lead to any mortality or treatment-related adverse signs. Over a 90-day sub-chronic period of repeated oral administration of NMN-C® at doses of 375, 750 and 1500 mg/kg/d followed by a 28-day treatment-free recovery period, NMN-C® appeared to be safe and did not promote toxic effects as seen from body weight change, food and water consumption, feed conversion efficiency, biochemical and blood parameters as well as organ toxicity and histological examinations of main organs. In conclusion, we provide the first data highlighting the safety of short to intermediate term (sub-chronic) oral administration of NMN and our experimental results allowed to determine a No-Observable Adverse Effect Level (NOAEL) for NMN-C® to be ≥ 1500 mg/kg/d.

The effect of prolonged intense physical exercise of special forces volunteers on their plasma protein denaturation profile examined by differential scanning calorimetry.

The human blood plasma proteome profile has been an area of intensive investigation and differential scanning calorimetry (DSC) has come forward as a novel tool in analyzing plasma heat capacity changes to monitor various physiological responses in health and disease. This study used DSC to assess potential alterations in the plasma heat capacity profile of albumin and globulins during extremely demanding physical exercise. We monitored the changes in denaturation profiles of those plasma proteins for five consecutive days of an extraordinary exercise training schedule in 14 young male Special Forces volunteers, as well as after a 30-day recovery period. The major effect of the prolonged intense exercise was the continuous upward shift of the albumin peak by 2°-3°C on the initial days of exercise, with a tendency to plateau circa the 5th day of exercise. In addition, some redistribution of the denaturational enthalpy was observed upon exercise, where the globulins peak increased relative to the albumin peak. Noteworthy, the alterations in the plasma proteome denaturational profiles were not persistent, as virtually full recovery of the initial status was observed after 30 days of recovery. Our findings indicate that 5 days of exhaustive physical exercise of highly trained individuals enhanced the thermal stability of plasma albumin shifting its denaturational transition to higher temperatures. We surmise that these effects may be a result of increased blood oxygenation during the prolonged intense exercise and, consequently, of albumin oxidation as part of the overall adaptation mechanisms of the body to extreme physical and/or oxidative stress.

Opioid-Free Recovery from Bunionectomy with HTX-011, a Dual-Acting Local Anesthetic Combining Bupivacaine and Meloxicam, as the Foundation of Non-Opioid Multimodal Analgesia.

There is a need for postoperative pain control that minimizes or eliminates opioid use during the first 72 hours following surgery, when pain is most severe. HTX-011 is an extended-release, dual-acting local anesthetic that demonstrated superior 72-hour analgesia over standard of care bupivacaine hydrochloride (HCl) and saline placebo in a phase 3 bunionectomy study (EPOCH-1). Having shown HTX-011 monotherapy is superior to bupivacaine HCl in reducing postoperative pain intensity and opioid use, this follow-on study evaluated the safety and efficacy of HTX-011 as the foundation of a multimodal analgesia (MMA) regimen using over-the-counter medications recommended by practice guidelines for pain management. Following regional anesthesia administered as a lidocaine block, patients underwent unilateral bunionectomy with osteotomy and internal fixation. Prior to closure, HTX-011 (up to 60 mg bupivacaine/1.8 mg meloxicam) was applied without a needle. Patients received scheduled postoperative MMA alternating ibuprofen (600 mg) and acetaminophen (1 g) every 3 hours for 72 hours. Efficacy was assessed based on pain intensity (numeric rating scale [NRS; 0-10]) and consumption of opioid rescue medication (intravenous morphine milligram equivalents [MME]). Adverse event and vital sign monitoring, plus laboratory and wound healing assessments, were used to determine safety. Over the 72-hour assessment period following bunionectomy, mean pain scores were mild in severity (NRS <4) and 22/31 patients (71%) experienced no severe pain (NRS ≥7) with HTX-011 as the foundation of scheduled, non-opioid MMA. Patients consumed an average of 1.61 MME total, with 24/31 (77%) requiring no opioid rescue medication (opioid-free). HTX-011 was well-tolerated and demonstrated no safety concerns with the inclusion of postoperative MMA. HTX-011 as the foundation of an MMA regimen including scheduled ibuprofen and acetaminophen maintained mean postoperative pain scores in the mild range and enabled opioid-free recovery for 77% of bunionectomy patients through the 28-day recovery period.

Sex-dependent long-term effects of prepubescent stress on the posterior parietal cortex

Adolescence is a time of intense cortical development and a period of heightened sensitivity to insult. To determine how sex affects the short- and long-term outcomes of early-adolescent stress exposure, we subjected prepubescent (postnatal day 30) male and female mice to repeated multiple concurrent stressors (RMS). In the posterior parietal cortex (PPC), RMS caused the elimination of excitatory synapses in deeper layers while inhibitory synapse density was predominantly diminished in superficial layers. These short-term effects coincided with reduced visuo-spatial working memory and were similar in both sexes. The loss of excitatory synapses and impaired working memory persisted in males past a 30-day recovery period. In contrast, we observed a remarkable recovery of excitatory transmission and behavioral performance in females. Inhibitory synapse density recovered in both sexes. We have also observed a late onset anxiety phenotype in RMS exposed females that was absent in males. Overall, our results indicate that there are marked sex differences in the long-term effects of prepubescent stress on cortical synapses and behavior.

Characterizing transcriptomic responses of southern flounder (Paralichthys lethostigma) chronically exposed to Deepwater Horizon oiled sediments

To obtain a deeper understanding of the transcriptomic responses to oil in southern flounder (Paralichthys lethostigma), we performed quantitative PCR and RNA sequencing on liver and gill tissue after a chronic exposure (35 days) to Deepwater Horizon crude oiled sediment and after a 30-day recovery period. We wanted to understand which specific genes are differentially expressed in liver and gill tissues directly after oiled sediment exposure and with the addition of a recovery period. Furthermore, we wanted to examine specific enriched pathways in these two tissues to determine the impact of exposure with and without a recovery period on biological processes (e.g. immune function). Liver and gill tissues were chosen because they represent two distinct organs that are highly important to consider when examining the impacts of oiled sediment exposure. The liver is the classic detoxification organ, while the gill is in direct contact with sediment in benthic fishes. Examination of these two tissues, therefore, generates a broad understanding of the transcriptomic consequences of oil exposure across an organism. Gene expression for interleukin 8 (il8) and interleukin 1B (il1β) was significantly increased versus control measurements for fish exposed to oiled sediments for 35 days in gill tissue. Hierarchical clustering of gene expression showed that tissue type was the main driver of gene expression (rather than treatment). The inclusion of a 30-day post-exposure recovery period showed a return of il8 and il1β gene expression in the gill to baseline expression levels. However, the recovery period increased the number of differentially expressed genes and significantly affected canonical pathways in both tissue types. Pathways related to cholesterol biosynthesis were significantly suppressed in oil-exposed flounder with a recovery period, but not in the exposed flounder without a recovery period. At the end of the exposure, 17 pathways were significantly affected in the gill, including thyroid hormone metabolism-related pathways, which were the most influenced. Liver tissue from the recovered fish had the greatest number of enriched pathways for any tissue or time point (187). Cellular and humoral immune response pathways were considerably impacted in the liver after the recovery period, suggesting that the immune system was attempting to respond to potential damage caused from the chronic oil exposure. Our results demonstrate that liver and gill tissues from southern flounder were differentially altered by Deepwater Horizon oiled sediment exposure and that a 30-day recovery period after exposure substantially shifted gene expression and canonical pathway profiles.

The effect of chronic exposure to chloridazon and its degradation product chloridazon-desphenyl on signal crayfish Pacifastacus leniusculus

The effects of chloridazon (Ch) and its metabolite chloridazon-desphenyl (Ch-D) at the environmentally relevant concentrations of 0.45 µg/L and 2.7 µg/L on signal crayfish Pacifastacus leniusculus were assessed in a 30-day exposure followed by a 15-day depuration period. Locomotion, biochemical haemolymph profile, oxidative and antioxidant parameters, and histopathology were evaluated. Crayfish exposed to Ch at 0.45 µg/L and 2.7 µg/L showed significantly (p < 0.01) higher CAT activity and GSH level in hepatopancreas and gill compared to controls. The concentration of Ch at 2.7 µg/L was associated with significantly (p < 0.01) higher levels of GLU, LACT, ALT, AST in haemolymph compared to controls. Chloridazon-desphenyl exposure at both tested concentrations caused significantly higher (p < 0.01) GLU, LACT, ALT, AST, NH3, and Ca in haemolymph; lipid peroxidation (TBARS) levels in hepatopancreas; and CAT activity and GSH level in hepatopancreas and gill. Alterations of structure including focal dilatation of tubules, increased number of fibrillar cells, and haemocyte infiltration in the interstitium were observed with 2.7 µg/L Ch and with both Ch-D exposures. Locomotion patterns did not vary significantly among groups. A 15-day recovery period was insufficient to restore normal physiological parameters in exposed groups. Chloridazon and its metabolite Ch-D exerts harmful effects on crayfish.

Genotoxicity and subchronic toxicity studies of supercritical carbon dioxide and acetone extracts of rosemary

Toxicology studies conducted with oil-soluble rosemary extracts to support authorization as a food additive (antioxidant) in the EU include an Ames test using a supercritical carbon dioxide extract (D74), a full 90-day study using D74 and an acetone extract (F62), and an investigative 90-day study with a 28-day recovery period (using D74 only). D74 was non-mutagenic in the Ames test. In the full 90-day study, where rats (20/sex/group) were either provided control diet or diets containing D74 (300, 600, or 2400 mg/kg) or F62 (3800 mg/kg), liver enlargement and hepatocellular hypertrophy were observed. To determine a mode of action and assess the reversibility of the hepatic effects, an investigative 90-day study was conducted using female rats (10/group receiving control diet or diet containing 2400 mg/kg D74). Liver enlargement was fully reversible after 28 days and microsomal enzyme analysis revealed reversible induction of cytochrome P450 enzymes (CYP2A1, CYP2A2, CYP2C11, CYP2E1, and CYP4A), demonstrating that the hepatic effects were adaptive and of no toxicological concern. Therefore, the highest dietary concentrations were established as the NOAELs. The investigative 90-day study NOAEL (providing 64 mg/kg bw/day carnosol and carnosic acid [the primary antioxidant components]) was used to establish a temporary ADI for rosemary extracts.

Evaluation of chronic toxicity of cyclocreatine, a creatine analog, in Sprague Dawley rat after oral gavage administration for up to 26 weeks

Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 μg/mL; AUC0-24 of 1970 h*μg/mL) was considered the maximum tolerated dose (MTD) in SD rats.

Identification of compound causing yellow bone discoloration following alpha-glycosyl isoquercitrin exposure in Sprague\u2013Dawley rats

Previous rat toxicity studies of alpha-glycosyl isoquercitrin (AGIQ), a water-soluble flavonol glycoside derived from rutin, revealed systemic yellow bone discoloration. This investigative study was conducted to determine the AGIQ metabolite(s) responsible for the discoloration. Female Sprague–Dawley rats were administered dietary AGIQ at doses of 0%, 1.5%, 3.0%, or 5.0% (0, 1735.0, 3480.8, and 5873.7 mg/kg/day, respectively) for 14 days, followed by a 14- or 28-day recovery period. Measurements of quercetin in urine and quercetin, quercetin 3-O-glucuronide, kaempferol, and 3-o-methylquercetin metabolites of AGIQ in bone (femur), white and brown fat, and cerebrum samples were conducted following the exposure period and each recovery period. Gross examination of the femur revealed yellow discoloration that increased in intensity with dose and was still present in a dose-related manner following both recovery periods. Quercetin, at levels correlating with AGIQ dose, was measured in the urine following the 14-day exposure period and, at lower concentrations, 14 or 28 days following cessation of AGIQ exposure. All four metabolites were present in a dose-dependent manner in the femur following 14 days of dietary exposure; only quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were present during the recovery periods. Quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were detected in white fat (along with kaempferol), brown fat (excluding quercetin due to analytical interference), and cerebrum samples, indicating systemic availability of the metabolites. Collectively, these data implicate quercetin, quercetin 3-O-glucuronide, or 3-o-methylquercetin (or a combination thereof) as the most likely metabolite of AGIQ causing the yellow discoloration of bone in rats administered dietary AGIQ.

Nitric oxide synthase-mediated sub-chronic injury and recovery in the small intestine of mice after oral administration with halloysite nanotubes.

Natural halloysite nanotubes (HNTs) with a hollow lumen have been widely applied in many fields, such as water purification, drug carriers, cosmetics, antibacterial, and scaffolds for tissue engineering. However, their in vivo toxicity is still largely unclear. The aim of this study is to evaluate sub-chronic oral toxicity of HNTs in the small intestine of mice. The results demonstrated that oral HNTs at low dose (5mg/kg) for 30days promoted mouse growth with no obvious adverse effect on the small intestine. The promotive effect on mouse growth disappeared after cessation of oral administration of HNTs. Oral HNTs at high dose (50mg/kg) for 30days induced aluminum (Al) and silicon (Si) accumulation and oxidative stress in the small intestine, which caused significant increases in the levels of cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) and inflammatory response and iNOS-mediated damages in the organ. Oral HNTs-induced changes in the small intestine at high dose were not observed after a 30-day recovery period. These findings provided the first evidence that oral HNTs-induced sub-chronic toxicity in the small intestine was reversible. The results suggest that HNTs at low concentration in environments have no adverse effect on mice, while there are health risks to mice under severe contamination by HNTs.

Testing the Limits of Sex Differences Using Variable Stress

Depression is a chronic disease that affects nearly twice as many women as men, and symptoms can differ by sex. Preclinical models disproportionately use male subjects and test a single behavioral endpoint immediately at the cessation of stress. We conducted variable stress in male and female mice for 6, 28, and 56 days, and measured behavior with a battery chosen to match research domain criteria. To examine individual differences, we generated a composite z score to measure stress susceptibility across behavioral tests. We also tested behavior following a 30-day recovery period to evaluate the duration of the stress effects. Females, but not males, were susceptible to 6 days of variable stress when behavioral testing started 24 h later. If behavioral testing was conducted 30 days later both males and females expressed stress related behaviors. Males and females were stress susceptible to 28 days of variable stress and effects were long lasting. Both sexes habituated to 56 days of variable stress, but anxiety associated measures still showed persistence. Performance on specific behavioral tests was often different between individuals and between sexes, and not all stressed animals were susceptible to all tested behaviors. These studies confirm that behavioral sex differences are detected in response to variable stress, and reveal information about individual differences. Use of a test battery that measures varying endophenotypes can be combined into a single stress susceptibility score as a tool similar to the scales/inventories used for the study of depression in humans. We present these data with the goal of furthering the field’s understanding sex differences and how they shape the biology of mood disorders.

Safety Assessment of a Hemp Extract using Genotoxicity and Oral Repeat-Dose Toxicity Studies in Sprague-Dawley Rats

Cannabinoids are extracted from Cannabis sativa L. and are used for a variety of medicinal purposes. Recently, there has been a focus on the cannabinoid Cannabidiol (CBD) and its potential benefits. This study investigated the safety of a proprietary extract of C. sativa, consisting of 9% hemp extract (of which 6.27% is CBD) and 91% olive oil. The mutagenic potential of the hemp extract was evaluated with the AMES assay inclusive of a hepatic drug metabolizing mix (S9) rich in CYP enzymes. The test article did not elicit evidence of bacterial mutagenicity. GLP compliant 14-day and a 90-day toxicity study were conducted. Olive oil was used as a control. The 90-day study had a 28-day recovery period. Treatments for the 14-day non-recovery range-finding study were 0, 1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14 days. There was a non-statistically significant (p > 0.05) decrease in body weights for the male and female rats receiving the test article. Hypoactivity, hyperactivity, reduced food consumption and piloerection were observed in the rats receiving 4000 mg test article/kg bw. Histopathology showed an increase in the size of liver cells (hypertrophy) around the central vein (centrilobular) in Groups 3 (3/10) and 4 (5/10) that correlated with increased liver weights. In the 90-day study, 8 groups of rats were dosed with 0, 200, 400 and 800 mg test article/kg bw/day. Groups 5 to 8 had a 28-day recovery. There were no test article-linked changes in clinical observations, physical examinations, Functional Observation Battery, ophthalmology, Motor Activity Assessment, hematology, clinical chemistries and macropathology (all groups). With the exception of the liver and adrenal gland, no test article-linked pathology was observed. For all rats receiving the test article, histopathology showed hypertrophy of liver cells around the central vein. The increase of liver weight is most likely caused by hypertrophy due to up-regulation of the hepatic drug metabolizing enzymes. The hepatocellular hypertrophy was completely reversed in 28 days and was not considered to be an adverse effect. Vacuolization of the adrenal zona fasciculata was observed in the control and 800 mg test article/kg bw groups. The vacuolization of the zona fasciculata was of the same incidence and severity in treatment and control male rats and correlated with an increased in the weights of the adrenal glands. In addition, a statistically significant increase (p<0.05) in adrenal-to-body weight ratios was observed for females receiving 800 mg test article/kg bw. This increase in adrenal-to-body weight ratio did not correlate with any of the pathology findings. The NOAEL for the test article is 800 mg/kg bw/day for female and 400 mg/kg bw/day for male Sprague Dawley rats.

Histopathological and histochemical effects of silver nanoparticles on the gills and muscles of African catfish (Clarias garepinus)

This study investigates the effects of silver nanoparticles (AgNPs) and silver nitrate (AgNO3) on gills and muscles in African catfish (Clarias garepinus). Changes in bioaccumulation, histopathological, and histochemical were used as biomarkers. The effects of AgNPs were recorded in some of these properties based on their size (20 and 40 nm) and their concentration (10 and 100 µg/L). The concentrations of silver (Ag) in gills and muscles tissues showed significant variations in all exposed groups compared to the control group. At tissue and cell levels, histopathological changes were observed. These changes include aneurism, collapse, subepithelial edema, epithelium lifting and hyperplasia of interlammellar epithelium, curling of secondary lamellae, epithelial cell necrosis, hypertrophy and proliferation of erythrocytes, bifurcation and fusion of filament, an increase in the number and size of mucous cells in the gills tissues. In addition, degeneration of muscle bundles, infiltration of inflammatory cells, focal areas of necrosis, splitting of muscle fibers, broken myofibrils, thickening of muscle bundles, dislocated striated muscles and shortening of muscle bundles in muscle tissues of fish exposed to AgNPs were exporated to evaluate the changes. Also, depletion of carbohydrates of the gill tissues and the muscle fibers with an increase in the number and size of mucous cells that condense in the secondary lamellae were observed. The 15-day recovery period improved most of the modifications in histopathological and histochemical parameters caused by AgNPs and AgNO3. In conclusion, one can assume the sensitivity of the gills and muscles of C. garepinus to AgNPs and recovery strategy is a must.