Afidopyropen: Challenges and impact of a toxicokinetic study designed to identify a point of inflection from dose-proportionality.

Afidopyropen is an insecticide that acts as a transient receptor potential vanilloid subtype (TRPV) channel modulator in chordotonal organs of target insects and has been assessed for a wide range of toxicity endpoints including chronic toxicity and carcinogenicity in rats and mice. The current study evaluates the toxicokinetic properties of afidopyropen and its plasma metabolites in rats at dose levels where the pharmacokinetics (PK) are linear and nonlinear in an attempt to identify a point of inflection. Based on the results of this study and depending on the analysis method used, the kinetically derived maximum dose (KMD) is estimated to be between 2.5 and 12.5 mg/kg bw/d with linearity observed at doses below 2.5 mg/kg bw/d. A defined point of inflection could not be determined. These data demonstrate that consideration of PK is critical for improving the dose-selection in toxicity studies as well as to enhance human relevance of the interpretation of animal toxicity studies. The study also demonstrates the technical difficulty in obtaining a defined point of inflection from in vivo PK data.

Dual Color Imaging from a Single BF2-Azadipyrromethene Fluorophore Demonstrated in vivo for Lymph Node Identification.

Dual emissions at ~700 and 800 nm have been achieved from a single NIR-AZA fluorophore 1 by establishing parameters in which it can exist in either its isolated molecular or aggregated states. Dual near infrared (NIR) fluorescence color lymph node (LN) mapping with 1 was achieved in a large-animal porcine model, with injection site, channels and nodes all detectable at both 700 and 800 nm using a preclinical open camera system. The fluorophore was also compatible with imaging using two clinical instruments for fluorescence guided surgery. Methods: An NIR-AZA fluorophore with hydrophilic and phobic features was synthesised in a straightforward manner and its aggregation properties characterised spectroscopically and by TEM imaging. Toxicity was assessed in a rodent model and dual color fluorescence imaging evaluated by lymph node mapping in a large animal porcine models and in ex-vivo human tissue specimen. Results: Dual color fluorescence imaging has been achieved in the highly complex biomedical scenario of lymph node mapping. Emissions at 700 and 800 nm can be achieved from a single fluorophore by establishing molecular and aggregate forms. Fluorophore was compatible with clinical systems for fluorescence guided surgery and no toxicity was observed in high dosage testing. Conclusion: A new, biomedical compatible form of NIR-dual emission wavelength imaging has been established using a readily accessible fluorophore with significant scope for clinical translation.

Dermal toxicity, dermal irritation, and delayed contact sensitization evaluation of oil body linked oleosin-hEGF microgel emulsion via transdermal drug delivery for wound healing.

Objective: The expression of therapeutic proteins in plant oil body bioreactors has attracted much attention. But its safety is not yet clear. This article determines the risk of safety after using the drug. Methods: The oil body-linked oleosin-hEGF microgel emulsion (OBEME) was prepared by mixing the xanthan gum with suitable concentrations in an appropriate proportion. Skin irritation and sensitization reaction were investigated in rats and guinea pigs using OBEME as test article.Results: The OBEME did not produce dermal erythema/eschar or oedema responses. The dermal subacute and subchronic toxicity of OBEME were evaluated in accordance with OECD guidelines. Compared with the control group, the basic physical signs, such as weight, feed, drinking, excretion, and behaviour of experimental animals, were not abnormal. In addition, no abnormality was found in haematological parameters, biochemical indexes, relative organ weight, and histopathological observation of organs, and there was no significant difference compared with normal saline treatment group. Therefore, we conclude that OBEME has no toxic effects and is safe and reliable to be used for topical application.

Cadmium-Induced Testicular Toxicity in Mice: Subacute and Subchronic Route-Dependent Effects.

This study aimed to compare Cd exposure by intraperitoneal (i.p.) and oral routes, evaluating the testicular subacute and subchronic effects. Adult male mice were separated into three groups subdivided according to the experimental period (7 and 42 days after Cd exposure: subacute and subchronic effects, respectively): one group received water and two groups received CdCl2 (1.2 mg/kg i.p. and 24 mg/kg oral). The testicular concentration of essential minerals and Cd, activity of antioxidant enzymes and markers of oxidative stress, histology, and testicular histomorphometry were evaluated. The subacute effect of oral Cd showed reduced Fe concentration, while Ca and Cu increased in this route. The subchronic effect promoted decreasing in Mg in i.p. and oral routes, whereas Zn decreased only in the oral, and the Fe concentration did not change. SOD activity decreased in the oral subacute evaluation and in both pathways, i.p. and oral routes, in the subchronic evaluation, while GST activity increased, and MDA concentration decreased. Labeling of apoptotic cells was increased in the subacute and subchronic evaluation. Seminiferous epithelium degeneration, death of germ cells, and Leydig cell damages occurred in i.p. and oral routes. However, these damages were more intense in the oral route, mainly evaluating the subchronic effects. The results confirm that the severity of Cd-induced testicular injury depends on the pathway, as well as the duration of exposure.

Relevant dose of the environmental contaminant, tributyltin, promotes histomorphological changes in the thyroid gland of male rats.

Organotin compounds, such as tributyltin (TBT), are common environmental contaminants and suspected endocrine-disrupting chemicals. Tributyltin is found in antifouling paints, widely used in ships and other vessels. The present study evaluated whether a 15-day treatment with TBT at a dose of 100 ng/kg/day could induce histomorphological changes in the thyroid gland of rats. TBT promoted relevant alterations in the thyroid architecture, being the most relevant histological findings the presence of increased number of small-size follicles in the treated group. In qualitative analyses, colloid vacuolization, papillary budging structures, cystic degeneration and chronic thyroiditis, were observed. Moreover, histomorphometric analysis showed statistically significant changes in the follicular architecture of TBT-treated rats, mainly a decrease in the follicle area (colloid) and an increased epithelial height that resulted in an increased epithelial height/colloid ratio. Augmented collagen deposition was also seen in the thyroids of treated groups. In immunohistochemical (IHC) analyses, the localization of NIS protein was described and a significant increased proliferation index (evaluated by Ki67 positive cells) in the treated group was reported. As an indirect measurement of oxidative stress, mitochondrial protein SDHA was also analyzed by IHC analysis. Although the cytoplasmic expression of SDHA was observed in both groups, the staining intensity score was higher in TBT-treated group. Our results suggest that besides causing histomorphological changes, environmental relevant dose of TBT treatment can also induce oxidative alterations.

Chronic dietary toxicity and carcinogenicity studies of dammar resin in F344 rats.

Dammar resin is a natural food additive and flavoring substance present in many foods and drinks. The present study evaluates the chronic toxicity and carcinogenicity of dietary dammar resin in F344 rats. Dietary concentrations in the 52-week chronic toxicity study were 0, 0.03, 0.125, 0.5, or 2%. The major treatment-related deleterious effects were body weight suppression, increased relative liver weight, and low hemoglobin levels in males and females. Foci of cellular alteration in the liver were observed in the male 2% group, but not in any other group. The no-observed-adverse-effect level for chronic toxicity was 0.125% for males (200.4 mg/kg b.w./day) and females (241.9 mg/kg b.w./day). Dietary concentrations in the 104-week carcinogenicity study were 0, 0.03, 0.5, or 2%. Dammar resin induced hemorrhagic diathesis in males and females, possibly via the inhibition of extrinsic and intrinsic coagulation pathways. Incidences of hepatocellular adenomas and carcinomas were significantly increased in the male 2% group, but not in any other group. In the 4-week subacute toxicity study, the livers of male rat-fed diet-containing 2% dammar resin had increased levels of protein oxidation and increased the expression of two anti-apoptotic and seven cytochrome P450 (CYP) genes. There was also an increased tendency of oxidative DNA damage. These findings demonstrate that dammar resin is hepatocarcinogenic in male F344 rats and underlines the roles of inhibition of apoptosis, induction of CYP enzymes, and oxidative stress in dammar resin-induced hepatocarcinogenesis.

Rac1-mediated cardiac damage causes diastolic dysfunction in a mouse model of subacute doxorubicin-induced cardiotoxicity.

The anticancer efficacy of anthracyclines is limited by congestive heart failure. Clinically established markers of early onset of cardiotoxicity following anthracycline treatment and preventive measures are missing. Although statins are reported to alleviate anthracycline-induced cardiotoxicity in vivo, the molecular mechanisms involved remain elusive. In vitro data point to Rac1 as major target of the cytoprotective statin effects. Here we investigated whether specific inhibition of Rac1 by NSC23766 is as effective as lovastatin in preventing subacute cardiotoxicity following doxorubicin treatment. C57BL/6 mice were treated over 3 weeks with multiple low doses of doxorubicin (6 × 3 mg/kg BW, i.p.) and the level of DNA damage, apoptosis and regenerative proliferation as well as pro-inflammatory, pro-fibrotic and oxidative stress responses were investigated. Moreover, heart function was monitored by echocardiography. Doxorubicin induced subacute cardiotoxicity which was reflected on the level of residual DNA damage, frequency of apoptotic and mitotic cells as well as elevated mRNA expression of markers of heart failure, remodeling and mitochondrial biogenesis. These molecular markers of cardiotoxicity were mitigated to a similar extent by co-treatment with either lovastatin (10 mg/kg BW, p.o.) or NSC23766 (5 mg/kg BW, i.p.) three times a week. Moreover, doxorubicin caused diastolic dysfunction as reflected by increased E-wave acceleration time (EAT), which again was prevented by pharmacological inhibition of Rac1. Inhibition of Rac1 signaling is of major relevance for the cardioprotective effects of lovastatin in the context of anthracycline-induced cardiotoxicity. Moreover, EAT is a useful marker of subacute cardiotoxicity caused by persisting harmful effects of doxorubicin.

Safety evaluation of auraptene in rats in acute and subacute toxicity studies.

Auraptene (AUR) is a natural, bioactive, monoterpene coumarin ether. It has anti-inflammatory, anti-carcinogenic, anti-bacterial, neuroprotective, and hepatoprotective properties. The aim of the present study was to assess the acute and subacute toxicity of oral administration of AUR in rats by evaluating clinical signs, haematology, biochemical factors, pathological changes and immune-toxicity. Acute administration of AUR in doses of 125, 250, 500, 1000 and 2000 mg/kg body weight had no mortality or clinical signs in a period of two days. To evaluate subacute toxicity, AUR was administrated for 28 days by oral gavage in doses of 125 and 250 mg/kg. There were significant differences in the haematological and biochemical data of the treated and untreated groups. However, almost all haematological differences were within normal reference ranges. Subacute administration of AUR showed no toxic histopathological effects on organ tissue. Evaluation of immune-toxicity also revealed no significant differences between treatment and untreated groups.

Protective effect of Devosia sp. ANSB714 on growth performance, serum chemistry, immunity function and residues in kidneys of mice exposed to deoxynivalenol.

This study was conducted to investigate the toxic effects of deoxynivalenol (DON) and the ameliorating efficacy of Devosia sp. ANSB714 for the negative effects of DON on mice. In the experiment, 80 mice were randomly divided into 4 treatments: non-toxin control, toxin, non-toxin control + ANSB714 and toxin + ANSB714. During 28 days, the mice in treatment with 4.70 mg/kg DON only had significantly lower average daily gain as compared those with non-toxin control treatment (P < 0.05). Serum blood urea nitrogen, tumour necrosis factor-α and the residues of DON in kidneys in mice received the toxin diet were obviously higher than those with non-toxin control (P < 0.05). There were no significant differences (P > 0.05) between ANSB714 treatments and non-ANSB714 treatments on above parameters of mice. Adding ANSB714 to toxic diets could normalize deviant physiological effects of DON on mice.

The transferability from rat subacute 4-week oral toxicity study to translational research exemplified by two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties.

Exposure to chemicals may have an influence on the immune system. Often, this is an unwanted effect but in some pharmaceuticals, it is the intended mechanism of action. Immune function tests and in depth histopathological investigations of immune organs were integrated in rodent toxicity studies performed according to an extended OECD test guideline 407 protocol. Exemplified by two immunosuppressive drugs, azathioprine and cyclosporine A, and two environmental chemicals, hexachlorobenzene and benzo[a]pyrene, results of subacute rat studies were compared to knowledge in other species particular in humans. Although immune function has a high concordance in mammalian species, regarding the transferability from rodents to humans various factors have to be taken into account. In rats, sensitivity seems to depend on factors such as strain, sex, stress levels as well as metabolism. The two immunosuppressive drugs showed a high similarity of effects in animals and humans as the immune system was the most sensitive target in both. Hexachlorobenzene gave an inconsistent pattern of effects when considering the immune system of different species. In some species pronounced inflammation was observed, whereas in primates liver toxicity seemed more obvious. Generally, the immune system was not the most sensitive target in hexachlorobenzene-treatment. Immune function tests in rats gave evidence of a reaction to systemic inflammation rather than a direct impact on immune cells. Data from humans are likewise equivocal. In the case of benzo[a]pyrene, the immune system was the most sensitive target in rats. In the in vitro plaque forming cell assay (Mishell-Dutton culture) a direct comparison of cells from different species including rat and human was possible and showed similar reactions. The doses in the rat study had, however, no realistic relation to human exposure, which occurs exclusively in mixtures and in a much lower range. In summary, a case by case approach is necessary when testing immunotoxicity. Improvements for the translation from animals to humans related to immune cells can be expected from in vitro tests which offer direct comparison with reactions of human immune cells. This may lead to a better understanding of results and variations seen in animal studies.

Acute and subacute toxicity studies on triptolide and triptolide-loaded polymeric micelles following intravenous administration in rodents.

Except its anti-tumour effects, triptolide (TP) also shows multiple pharmacological side activities, such as immune-suppressive and male anti-fertility. To increase the therapeutic index of TP, a novel polymeric micelle system containing TP (TP-PM) has been developed to treat tumour. Our previous studies have demonstrated the good anti-tumour efficacy of TP-PM. This paper investigated the acute toxicity in mice and subacute toxicity in rats of TP-PM and TP. Results demonstrated that the LD50 for TP-PM and TP administered intravenously were 1.06 mg/kg and 0.83 mg/kg in mice, respectively. In subacute toxicity study, TP-PM and TP were administered intravenously at the dose levels of 0.1 mg/kg and 0.3 mg/kg for 14 d. Compared to the control, there was significant decrease in the serum AST activities, the testis ACP activities, thymus index, testis index, and significant increase in spleen index, and obvious histopathological changes in rats treated with TP, however, the toxicities of TP-PM on liver, kidney, testis and spleen are slighter than TP. Compared to TP, TP-PM significantly increased the ACP activity of the testis and decreased the MDA level in serum. So, the polymeric micelles may be a novel drug delivery carrier of TP for reducing the toxicities of TP.

Evaluation of novel acute urinary rat kidney toxicity biomarker for subacute toxicity studies in preclinical trials.

Novel urinary protein biomarkers for the detection of acute renal damage, recently accepted by the U.S. Food and Drug Administration, European Medicines Agency, and Pharmaceuticals and Medical Devices Agency (Japan), now have to be validated in practice. Limited data regarding the performance of these acute markers after subacute or subchronic treatment are publicly available. To increase the area of applicability of these markers, it is important to evaluate the ability to detect them after 28 days of treatment or even longer. Wistar rats were treated with three doses of cisplatin, vancomycin, or puromycin to induce renal damage. Twelve candidate proteins were measured by Luminex xMAP-based WideScreen assays, MesoScale Discovery-based MULTI-SPOT technology, or RENA-strip dipstick assay after 28 days. Treatment with all three model compounds resulted in a dose-dependent increase in urinary biomarkers, specific for the observed areas within the nephron, determined histopathologically. The most promising biomarkers in this study were NGAL, Kim-1, osteopontin, clusterin, RPA-1, and GSTYb1, detected by multiplexing technologies. The RENA-strip dipstick assay delivered good diagnostic results for vancomycin-treated but not for cisplatin- or puromycin-treated rats. Taken together, the data show that these new biomarkers are robust and measurable for longer term studies to predict different types of kidney toxicities.