Safety evaluation of a novel topical combination of esafoxolaner, eprinomectin and praziquantel, in reproducing female cats.

NexGard® Combo, a novel topical endectoparasiticide product for cats, is a combination of esafoxolaner, eprinomectin and praziquantel. The safety of this novel combination administered to females during reproduction and lactation was evaluated per analysis of breeding parameters and adverse reactions observed on females and offspring. Females with successful breeding history were randomized to three groups, a placebo group and groups treated with the novel formulation at 1× or 3× multiples of the maximum exposure dose. Females were dosed at 28-day intervals, at least twice before mating, then during a period including mating, pregnancy, whelping and 56 days of lactation. In the placebo, 1× and 3× groups, 10, 9 and 10 females, respectively completed the study (nine, seven and nine females achieved pregnancy), and were dosed 7.1 times on average. Breeding parameters included success of mating, success of gestation, length of gestation, abortion rate, number of live, dead and stillborn kittens at birth, number of kittens with abnormalities, weight of kittens after birth and at weaning, growth of kittens, proportion of male and female kittens, and proportion of kittens born alive and weaned. No significant adverse reactions related to the novel combination were observed on females and on kittens; no significant and adverse effects on breeding parameters were observed.

TITLE: Évaluation de l'innocuité d'une nouvelle combinaison topique d'esafoxolaner, d'éprinomectine et de praziquantel chez les chattes reproductrices. ABSTRACT: NexGard® Combo, un nouvel endectoparasiticide topique pour chats, est une combinaison d'esafoxolaner, d'éprinomectine et de praziquantel. La sécurité de cette nouvelle association administrée aux chattes pendant la reproduction et la lactation a été évaluée par analyse des paramètres d'élevage et des effets indésirables observés sur les femelles et les descendants. Les chattes ayant des antécédents de reproduction réussie ont été randomisées en trois groupes, un groupe placebo et des groupes traités avec la nouvelle formulation à des multiples de 1× ou 3× la dose d'exposition maximale. Les femelles ont reçu des doses à 28 jours d'intervalle, au moins deux fois avant l'accouplement, puis pendant une période comprenant l'accouplement, la gestation, la mise bas et 56 jours de lactation. Dans les groupes placebo, 1× et 3×, repectivement dix, neuf et dix chattes ont terminé l'étude (neuf, sept et neuf chattes ont été gestantes) et ont été traitées 7,1 fois en moyenne. Les paramètres d'élevage comprenaient le succès de l'accouplement, le succès de la gestation, la durée de la gestation, le taux d'avortement, le nombre de chatons vivants, morts et mort-nés à la naissance, le nombre de chatons présentant des anomalies, le poids des chatons après la naissance et au sevrage, la croissance des chatons, la proportion de chatons mâles et femelles et la proportion de chatons nés vivants et sevrés. Aucun effet indésirable significatif lié à la nouvelle association n'a été observé chez les femelles et les chatons et aucun effet indésirable significatif sur les paramètres d'élevage n'a été observé.

Pharmacokinetic and Genomic Effects of Arsenite in Drinking Water on Mouse Lung in a 30-Day Exposure.

The 2 objectives of this subchronic study were to determine the arsenite drinking water exposure dependent increases in female C3H mouse liver and lung tissue arsenicals and to characterize the dose response (to 0, 0.05, 0.25, 1, 10, and 85 ppm arsenite in drinking water for 30 days and a purified AIN-93M diet) for genomic mouse lung expression patterns. Mouse lungs were analyzed for inorganic arsenic, monomethylated, and dimethylated arsenicals by hydride generation atomic absorption spectroscopy. The total lung mean arsenical levels were 1.4, 22.5, 30.1, 50.9, 105.3, and 316.4 ng/g lung tissue after 0, 0.05, 0.25, 1, 10, and 85 ppm, respectively. At 85 ppm, the total mean lung arsenical levels increased 14-fold and 131-fold when compared to either the lowest noncontrol dose (0.05 ppm) or the control dose, respectively. We found that arsenic exposure elicited minimal numbers of differentially expressed genes (DEGs; 77, 38, 90, 87, and 87 DEGs) after 0.05, 0.25, 1, 10, and 85 ppm, respectively, which were associated with cardiovascular disease, development, differentiation, apoptosis, proliferation, and stress response. After 30 days of arsenite exposure, this study showed monotonic increases in mouse lung arsenical (total arsenic and dimethylarsinic acid) concentrations but no clear dose-related increases in DEG numbers.

Myeloperoxidase-induced genomic DNA-centered radicals.

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.

Genome-wide analysis of BEAS-2B cells exposed to trivalent arsenicals and dimethylthioarsinic acid.

Lung is a major target for arsenic carcinogenesis in humans by both oral and inhalation routes. However, the carcinogenic mode of action of arsenicals is unknown. We investigated the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsinous acid (DMAIII) and dimethylthioarsinic acid (DMTA), a sulfur containing dimethyl arsenic metabolite, in human bronchial epithelial (BEAS-2B) cells. Cells were exposed to 3, 15 microM-iAsIII; 0.3, 1 microM-MMAIII; 0.2, 1 microM-DMAIII; 0.2, 0.9 microM-DMTA as non-cytotoxic and minimally cytotoxic ( approximately 20%) concentrations based on Neutral Red uptake assays after 24h of culture. Total RNA was isolated and gene expression analysis conducted using Affymetrix Human Genome 133 Plus 2.0 arrays. Differentially expressed genes (DEGs) were determined using a one-way ANOVA (p < or =0.05) by Rosetta Resolver, a Benjamini-Hochberg FDR (false discovery rate) multiple testing correction (< 0.05) followed by a Scheffe's post hoc test. For all compounds except DMTA, > 90% of DEG altered in the low concentration were also changed at the high concentration. There was a clear dose-response seen in the number of DEGs for all four compounds. iAsIII showed the highest number of DEG at both concentrations (2708 and 123, high and low, respectively). 1749, 420 and 120 DEGs were unique to the high concentrations of iAsIII, MMAIII and DMAIII, respectively. Transferrin receptor is a common DEG in low concentration arsenical treated cells. Ingenuity Pathway Analysis revealed p53 signaling (E2F1 and 2, SERPIN), and cell cycle related genes (cyclin D1) were altered by the high concentrations of DMTA, MMAIII and iAsIII. Oxidative stress (DUSP1, GPX2, NQO1, GCLC) and NF-kappaB signaling (TLR4, NF-kappaB) pathways were changed by the high concentrations of MMAIII and iAsIII. The genes identified in this study can be a valuable tool to determine the mechanism of arsenic toxicity and cancer formation. A number of similarities were observed in the gene expression profiles of DMAIII and DMTA and also iAsIII and MMAIII. These findings reveal some biological effects of arsenicals that will aid in creating a better risk assessment model for arsenical-induced lung cancer.

Proteomics of S-(1, 2-dichlorovinyl)-L-cysteine-induced acute renal failure and autoprotection in mice.

Previous studies (Vaidya VS, Shankar K, Lock EA, Bucci TJ, Mehendale HM. Toxicol Sci 74: 215-227, 2003; Korrapati MC, Lock EA, Mehendale HM. Am J Physiol Renal Physiol 289: F175-F185, 2005; Korrapati MC, Chilakapati J, Lock EA, Latendresse JR, Warbritton A, Mehendale HM. Am J Physiol Renal Physiol 291: F439-F455, 2006) demonstrated that renal repair stimulated by a low dose of S-(1,2-dichlorovinyl)l-cysteine (DCVC; 15 mg/kg i.p.) 72 h before administration of a normally lethal dose (75 mg/kg i.p.) protects mice from acute renal failure (ARF) and death (autoprotection). The present study identified the proteins indicative of DCVC-induced ARF and autoprotection in male Swiss Webster mice. Renal dysfunction and injury were assessed by plasma creatinine and histopathology, respectively. Whole-kidney homogenates were run on two-dimensional gel electrophoresis gels, and the expression of 18 common proteins was maximally changed (> or =10-fold) in all the treatment groups and they were conclusively identified by liquid chromatography tandem mass spectrometry. These proteins were mildly downregulated after low dose alone and in autoprotected mice in contrast to severe downregulation with high dose alone. Glucose-regulated protein 75 and proteasome alpha-subunit type 1 were further investigated by immunohistochemistry for their localization in the kidneys of all the groups. These proteins were substantially higher in the proximal convoluted tubular epithelial cells in the low-dose and autoprotected groups compared with high-dose alone group. Proteins involved in energetics were downregulated in all the three groups of mice, leading to a compromise in cellular energy. However, energy is recovered completely in low-dose and autoprotected mice. This study provides the first report on proteomics of DCVC-induced ARF and autoprotection in mice and reflects the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicological paradigms.

Role of CYP2E1 and saturation kinetics in the bioactivation of thioacetamide: Effects of diet restriction and phenobarbital.

Thioacetamide (TA) undergoes saturation toxicokinetics in ad libitum (AL) fed rats. Diet restriction (DR) protects rats from lethal dose of TA despite increased bioactivation-mediated liver injury via CYP2E1 induction. While a low dose (50 mg TA/kg) produces 6-fold higher initial injury, a 12-fold higher dose produces delayed and mere 2.5-fold higher injury. The primary objective was to determine if this less-than-expected increase in injury is due to saturation toxicokinetics. Rats on AL and DR for 21 days received either 50 or 600 mg TA/kg i.p. T(1/2) and AUCs for TA and TA-S-oxide were consistent with saturable kinetics. Covalent binding of (14)C-TA-derived-radiolabel to liver macromolecules after low dose was 2-fold higher in DR than AL rats. However, following lethal dose, no differences were found between AL and DR. This lack of dose-dependent response appears to be due to saturation of bioactivation at the higher dose. The second objective was to investigate the effect of phenobarbital pretreatment (PB) on TA-initiated injury following a sub-lethal dose (500 mg/kg). PB induced CYP2B1/2 approximately 350-fold, but did not increase covalent binding of (14)C-TA, TA-induced liver injury and mortality, suggesting that CYP2B1/2 has no major role in TA bioactivation. The third objective was to investigate the role of CYP2E1 using cyp2e1 knockout mice (KO). Injury was assessed over time (0-48 h) in wild type (WT) and KO mice after LD(100) dose (500 mg/kg) in WT. While WT mice exhibited robust injury which progressed to death, KO mice exhibited neither initiation nor progression of injury. These findings confirm that CYP2E1 is responsible for TA bioactivation.

Toxicokinetics and toxicity of thioacetamide sulfoxide: a metabolite of thioacetamide.

Thioacetamide (TA) is bioactivated by CYP2E1 to TA sulfoxide (TASO), and to the highly reactive sulfdioxide (TASO(2)), which initiates hepatic necrosis by covalent binding. Previously, we have established that TA exhibits saturation toxicokinetics over a 12-fold dose range, which explains the lack of dose-response for bioactivation-based liver injury. In vivo and in vitro studies indicated that the second step (TASO-->TASO(2)) of TA bioactivation is less efficient than the first one (TA-->TASO). The objective of the present study was to specifically test the saturation of the second step of TA bioactivation by directly administering TASO, which obviates the contribution from first step, i.e. TA-->TASO. Male SD rats were injected with low (50mg/kg, ip), medium (100mg/kg) and high (LD(70), 200mg/kg) doses of TASO. Bioactivation-mediated liver injury that occurs in the initial time points (6 and 12h), estimated by plasma ALT, AST and liver histopathology over a time course, was not dose-proportional. Escalation of liver injury thereafter was dose dependent: low dose injury subsided; medium dose injury escalated upto 36h before declining; high dose injury escalated from 24h leading to 70% mortality. TASO was quantified in plasma by HPLC at various time points after administration of the three doses. With increasing dose (i.e., from 50 to 200mg/kg), area under the curve (AUC) and C(max) increased more than dose proportionately, indicating that TASO bioactivation exhibits saturable kinetics. Toxicokinetics and initiation of liver injury of TASO are similar to that of TA, although TASO-initiated injury occurs at lower doses. These findings indicate that bioactivation of TASO to its reactive metabolite is saturable in the rat as suggested by previous studies with TA.

Preplaced cell division: a critical mechanism of autoprotection against S-1,2-dichlorovinyl-L-cysteine-induced acute renal failure and death in mice.

Previous studies have shown that renal injury initiated by a lethal dose of S-1,2-dichlorovinyl-l-cysteine (DCVC) progresses due to inhibition of cell division and hence renal repair, leading to acute renal failure (ARF) and death in mice. Renal injury initiated by low to moderate doses of DCVC is repaired by timely and adequate stimulation of renal cell division, tubular repair, restoration of renal structure and function leading to survival of mice. Recent studies have established that mice primed with a low dose of DCVC (15 mg/kg i.p.) 72 h before administration of a normally lethal dose (75 mg/kg i.p.) are protected from ARF and death (nephro-autoprotection). We showed that renal cell division and tissue repair stimulated by the low dose are sustained even after the lethal dose administration resulting in survival from ARF and death. If renal cell division induced by the low dose is indeed the critical mechanism of this autoprotection, then its ablation by the antimitotic agent colchicine (1.5 mg CLC/kg i.p.) should abolish autoprotection. The present interventional experiments were designed to test the hypothesis that DCVC autoprotection is due to stimulated cell division and tissue repair by the priming low dose. CLC intervention at 42 and 66 h after the priming dose resulted in marked progressive elevation of plasma blood urea nitrogen and creatinine resulting in ARF and death of mice. Light microscopic examination of hematoxylin and eosin-stained kidney sections revealed progression of renal necrosis concordant with progressively failing renal function. With CLC intervention, S-phase stimulation (as assessed by BrdU pulse labeling), G(1)-to-S phase clearance, and cell division were diminished essentially abolishing the promitogenic effect of the priming low dose of DCVC. Phospho-retinoblastoma protein (P-pRB), a crucial protein for S-phase stimulation, and other cellular signaling mechanisms regulating P-pRB were investigated. We report that decreased P-pRB via activation of protein phosphatase-1 by CLC is the critical mechanism of this inhibited S-phase stimulation and ablation of autoprotection with CLC intervention. These findings lend additional support to the notion that stimulated cell division and renal tissue repair by the priming dose of DCVC are the critical mechanisms that allow sustained compensatory tissue repair and survival of mice in nephro-autoprotection.

Protective effect of type 2 diabetes on acetaminophen-induced hepatotoxicity in male Swiss-Webster mice.

Type 2 diabetic (DB) mice exposed to CCl(4) (LD(50) = 1.25 ml/kg), acetaminophen (LD(80) = 600 mg/kg; APAP), and bromobenzene (LD(80) = 0.5 ml/kg) i.p. yielded 30, 20, and 20% mortality, respectively, indicating hepatotoxic resistance. Male Swiss-Webster mice were made diabetic by feeding high fat and administrating streptozotocin (120 mg/kg i.p.) on day 60. On day 71, time-course studies after APAP (600 mg/kg) treatment revealed identical initial liver injury in non-DB and DB mice, which progressed only in non-DB mice, resulting in 80% mortality. The hypothesis that decreased APAP bioactivation, altered toxicokinetics, and/or increased tissue repair are the underlying mechanisms was investigated. High-performance liquid chromatography analysis revealed no difference in plasma and urinary APAP or detoxification of APAP via glucuronidation between DB and non-DB mice. Hepatic CYP2E1 protein and activity, glutathione, and [(14)C]APAP covalent binding did not differ between DB and non-DB mice, suggesting that lower bioactivation-based injury is not the mechanism of decreased hepatotoxicity in DB mice. Diabetes increased cells in S phase by 8-fold in normally quiescent liver of these mice. Immunohistochemistry revealed overexpression of calpastatin in the newly dividing/divided cells, explaining inhibition of hydrolytic enzyme calpain in perinecrotic areas and lower progression of APAP-initiated injury in the DB mice. Antimitotic intervention of diabetes-associated cell division with colchicine before APAP administration resulted in 70% mortality in APAP-treated colchicine-intervened DB mice. These studies suggest that advancement of cells in the cell division cycle and higher tissue repair protect DB mice by preventing progression of APAP-initiated liver injury that normally leads to mortality.

Saturation toxicokinetics of thioacetamide: role in initiation of liver injury.

Thioacetamide (TA), a potent centrilobular hepatotoxicant, undergoes a two-step bioactivation mediated by microsomal CYP2E1 to TA sulfoxide (TASO), and further to TA-S,S-dioxide (TASO2), a reactive metabolite that initiates cellular necrosis. Our earlier studies showed that bioactivation-mediated liver injury of TA is not dose-proportional. The objective of this study was to examine whether increasing doses of TA lead to enzyme saturation, thereby resulting in lack of dose-response for injury: bioactivation of TA --> TASO --> TASO2 may follow zero-order kinetics. A 12-fold dose range of TA (50, 300, and 600 mg/kg i.p.) was injected into male Sprague-Dawley rats. TA and TASO were quantified in plasma, liver, and urine by high-performance liquid chromatography. With increasing doses, the apparent elimination half-lives of TA and TASO increased linearly, indicating that TA bioactivation exhibits saturation kinetics. Increasing TA dose resulted in greater-than-proportional increases in plasma TA and TASO levels. The TASO/TA ratio was inversely proportional to the dose of TA. Covalent binding of 14C-TA-derived radiolabel to liver macromolecules showed a less-than-dose-proportionate increase with a 12-fold higher dose. Less than dose-proportional covalent binding was confirmed in liver microsomal incubations with 14C-TA. Three-fold higher excretion of TASO was seen in urine at the highest dose (600 mg/kg) compared with the lowest dose (50 mg TA/kg). Incubation of TA with rat liver microsomes and purified baculovirus-expressed rat and human CYP2E1 Supersomes, over a concentration range of 0.01 to 10 mM, revealed saturation of TA conversion to TASO at and above 0.05 mM TA concentration, comparable to in vivo plasma and liver levels achieved upon administration of higher doses. Calculated K(m) values for TA (0.1 mM) and TASO (0.6 mM) suggest that the second step of TA bioactivation is 6-fold less efficient. Collectively, the findings indicate saturation of CYP2E1 at the first (TA to TASO) and second (TASO to TASO2) steps of TA bioactivation.