Victor Castellano

Permethrin-induced oxidative stress and toxicity and metabolism. A review

Permethrin (PER), the most frequently used synthetic Type I pyrethroid insecticide, is widely used in the world because of its high activity as an insecticide and its low mammalian toxicity. It was originally believed that PER exhibited low toxicity on untargeted animals. However, as its use became more extensive worldwide, increasing evidence suggested that PER might have a variety of toxic effects on animals and humans alike, such as neurotoxicity, immunotoxicity, cardiotoxicity, hepatotoxicity, reproductive, genotoxic, and haematotoxic effects, digestive system toxicity, and cytotoxicity. A growing number of studies indicate that oxidative stress played critical roles in the various toxicities associated with PER. To date, almost no review has addressed the toxicity of PER correlated with oxidative stress. The focus of this article is primarily to summarise advances in the research associated with oxidative stress as a potential mechanism for PER-induced toxicity as well as its metabolism. This review summarises the research conducted over the past decade into the reactive oxygen species (ROS) generation and oxidative stress as a consequence of PER treatments, and ultimately their correlation with the toxicity and the metabolism of PER. The metabolism of PER involves various CYP450 enzymes, alcohol or aldehyde dehydrogenases for oxidation and the carboxylesterases for hydrolysis, through which oxidative stress might occur, and such metabolic factors are also reviewed. The protection of a variety of antioxidants against PER-induced toxicity is also discussed, in order to further understand the role of oxidative stress in PER-induced toxicity. This review will throw new light on the critical roles of oxidative stress in PER-induced toxicity, as well as on the blind spots that still exist in the understanding of PER metabolism, the cellular effects in terms of apoptosis and cell signaling pathways, and finally strategies to help to protect against its oxidative damage.

The role of in vitro methods as alternatives to animals in toxicity testing

Introduction: It is accepted that animal testing should be reduced, refined or replaced as far as it is practicably possible. There are also a wide variety of in vitro models, which are used as screening studies and mechanistic investigations. The ability of an in vitro assay to be reliable, biomedically, is essential in pharmaceutical development. Furthermore, it is necessary that cells used in in vitro testing mimic the phenotype of cells within the human target tissue. Areas covered: The focus of this review article is to identify the key points of in vitro assays. In doing so, the authors take into account the chemical agents that are assessed and the integrated in vitro testing strategies. Expert opinion: There is a transfer of toxicological data from primary in vivo animal studies to in vitro assays. The key element for designing an integrated in vitro testing strategy is summarized as follows: exposure modeling of chemical agents for in vitro testing; data gathering, sharing and read-across for testing a class of chemical; a battery of tests to assemble a broad spectrum of data on different mechanisms of action to predict toxic effects; and applicability of the test and the integrated in vitro testing strategies and flexibility to adjust the integrated in vitro testing strategies to test substance. While these methods will be invaluable if effective, more studies must be done to ensure reliability and suitability of these tests for humans.

Dermatofibroma parasitized by Leishmania in HIV infection: a new morphologic expression of dermal Kala Azar in an immunodepressed patient.

Visceral leishmaniasis is a protozoan infection that may complicate the course of patients with human immunodeficiency virus (HIV). Dermatofibroma is a cutaneous fibrohistiocytic lesion considered neoplastic by some authors and inflammatory by others. Eruptive dermatofibromas have been described in patients with HIV infection or with other altered immunity situations. We present the case of a 32‐year‐old, HIV‐positive man with visceral leishmaniasis who complained of the appearance of a cutaneous lesion in the leg formed by the coexistence of dermatofibroma and Leishmania parasitic colonization. As far as we know, this type of association has not been reported previously. We consider that the dermatofibroma could have developed as an unusual form of fibrohistiocytic reaction to leishmania. From a practical approach, we recommend the search of leishmaniasis in dermatofibroma in immunosupressed patients.

Mycotoxins modify the barrier function of Caco-2 cells through differential gene expression of specific claudin isoforms: Protective effect of illite mineral clay.

Aflatoxin B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA) and T-2 toxin (T2) are mycotoxins that commonly contaminate the food chain and cause various toxicological effects. Their global occurrence is regarded as an important risk factor for human and animal health. In this study, the results demonstrate that, in human Caco-2 cells, AFB1, FB1, OTA and T2 origin cytotoxic effects, determining cell viability through MTT assay and LDH leakage, and decrease trans-epithelial electrical resistance (TEER). The decrease in barrier properties is concomitant with a reduction in the expression levels of the tight junction constituents claudin-3, claudin-4 and occludin. The protective effect of mineral clays (diosmectite, montmorillonite and illite) on alterations in cell viability and epithelial barrier function induced by the mycotoxins was also evaluated. Illite was the best clay to prevent the mycotoxin effects. Illite plus mycotoxin co-treatment completely abolished AFB1 and FB1-induced cytotoxicity. Also, the decreases in the gene expression of claudins and the reduction of TEER induced by mycotoxins were reversed by the illite plus mycotoxin co-treatment. In conclusion, these results demonstrated that mycotoxins AFB1, FB1, T2 and OTA disrupt the intestinal barrier permeability by a mechanism involving reduction of claudin isoform expressions, and illite counteracts this disruption.

Plasma disposition and tissue depletion of difloxacin and its metabolite sarafloxacin in the food producing animals, chickens for fattening.

Chickens were used to investigate plasma disposition of difloxacin after single intravenous (IV) and oral dose (10 mg/kg body weight (BW)) and to study residue depletion of difloxacin and its major metabolite sarafloxacin after multiple oral doses (10 mg difloxacin/kg BW, daily for 5 days). Plasma and tissue samples were analyzed using a HPLC method. After IV and oral administration, plasma drug concentration-time curves were best described by a two-compartment open model. Mean (± SD) elimination half-lives (t(½)β) of difloxacin were 9.53±1.00 and 12.23±1.81 h after IV and oral administration. Maximum plasma concentration was 2.34±0.50 μg/ml and interval from oral administration until maximal concentration was 1.34±0.03 h. Oral bioavailability was found to be 68.89±15.21%. Difloxacin was converted to sarafloxacin. After multiple oral dose (10mg difloxacin/kg BW, daily for 5 days), mean kidney, liver, muscle and skin + fat tissue concentrations of difloxacin and sarafloxacin ranging between 604.8±132.5 and 368.1±52.5 μg/kg and 136.4±18.3 and 10.4±1.2 μg/kg, respectively, were measured 1 day after administration of the final dose of difloxacin. A withdrawal time of 5 days was necessary to ensure that the residues of difloxacin were less than the maximal residue limits (MRL) or tolerance established by the European Union.

Acute and Repeated Dose (28 Days) Oral Safety Studies of ALIBIRD in Rats

ALIBIRD, a test substance composed of oligosaccharides derived from lactulose, a hydrolysate of a whey protein concentrate, and a supercritical extract of rosemary (1:0.5:0.05), was prepared in the laboratory and evaluated for its safety as a multifunctional food additive. In oral toxicity studies (acute and 28 days repeated dose) using Wistar rats, ALIBIRD was administered in a single oral gavage dose of 2,000 mg/kg of body weight and resulted in no adverse events or mortality; a daily dose of 2,000 mg/kg of body weight for 28 days by gavage also resulted in no adverse effects or mortality. No abnormal clinical signs, behavioral changes, body weight changes, or changes in food and water consumption occurred in either study. There were no changes in hematological and serum chemistry values, organ weights, or gross or histological characteristics. Based on test results, it is concluded that ALIBIRD is well tolerated in rats at an acute and subchronic (28 days) dose of 2,000 mg/kg of body weight.

Evidence for dose-additive effects of a type II pyrethroid mixture. In vitro assessment.

Despite the widespread use of pyrethroid insecticides that led to common exposure in the population, few studies have been conducted to quantitatively assess dose-additive effects of pyrethroids using a funcional measure involved in the common toxic mode of action. The aim of this study was to evaluate the potency and efficacy of 6 Type II pyretroids (α-cypermethrin, cyfluthrin, λ-cyhalothrin, deltamethrin, cyphenothrin and esfenvalerate) to evoke induction of both nitric oxide and lipid peroxides levels measured as malondialdehyde in three in vitro models (SH-SY5Y, HepG2 and Caco-2 human cells) as well as to test the hypothesis of dose additivity for mixtures of these same 6 pyrethroids. Concentration-responses for 6 pyrethroids were determined as well as the response to mixtures of all 6 pyrethroids. Additivity was tested assuming a dose-additive model. The human neuroblastoma SH-SY5Y cell line was the most sensitive in vitro model. The rank order of potency for cell SH-SY5Y viability MTT assay was deltamethrin>cyphenothrin>λ-cyhalothrin>cyfluthrin>esfenvalerate>α-cypermethrin. When 6 pyrethroids were present in the mixture at an equitoxic mixing ratio, the action on nitric oxide (NO) and lipid peroxides measured as malondialdehyde (MDA) production was consistent with a dose-additive model. The results of the present study are consistent with previous reports of additivity of pyrethroids in vivo e in vitro.

Differential induction of cytochrome P450 isoforms and peroxisomal proliferation by cyfluthrin in male Wistar rats

Cyfluthrin effects on in vivo drug metabolizing enzymes were evaluated using the oxidative substrate antipyrine. Antipyrine pharmacokinetics in plasma and urinary excretion of its major metabolites with and without cyfluthrin oral treatment (20mg/kg/day for 6 days) were investigated in rats. Cyfluthrin increased the apparent intrinsic clearance and decreased the antipyrine half-life at β phase. Cyfluthrin also increased the clearance of the antipyrine metabolites, norantipyrine, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine and the formation rate constants for each of the three metabolites measured in urine. These results suggest that cyfluthrin affects hepatic cytochrome P450 (CYP) system. In order to confirm, a second experiment was carried out. We evaluated the effects of repeated exposure to cyfluthrin on hepatic and renal CYP2E, CYP1A and CYP4A subfamilies and peroxisomal proliferation in rats following oral administration (10 and 20mg/kg/day for 6 days). At the highest dose, cyfluthrin increased renal and hepatic O-deethylation of ethoxyresorufin and O-demethylation of methoxyresorufin, metabolism mediated by the CYP1A subfamily. Liver and kidney were susceptible to cyfluthrin-dependent induction of 12- and 11-hydroxylation of lauric acid, suggesting CYP4A subfamily induction. Also cyfluthrin increased the β-oxidation of palmitoyl-coenzyme A and carnitine acetyltransferase activity, supporting cyfluthrin as a peroxisome proliferator. In conclusion, the demonstration that cyfluthrin induced hepatic CYP1A, CYP4A subfamilies and peroxisomal proliferation raises the possibility of cyfluthrin could produce changes in oxidative stress.

Fipronil sulfone induced higher cytotoxicity than fipronil in SH-SY5Y cells: Protection by antioxidants.

Fipronil is a broad spectrum insecticide from the phenyl pyrazole family, which targets GABA receptor. Limited information is available about the metabolite fipronil sulfone cytotoxic actions. This study examined in vitro neurotoxicity of fipronil and fipronil sulfone and evaluated Trolox (vitamin E analog) (0.3, 1μM), N-acetyl-cysteine (0.5, 1mM), melatonin (0.1, 1μM) and Tempol (superoxide dismutase analog) (0.3, 0.5mM) protective role in SH-SY5Y cells. MTT and LDH assays were carried out to assess the cytotoxicity of fipronil and fipronil sulfone at 3-100μM concentrations. Fipronil sulfone was more toxic than fipronil. Tempol showed the best neuroprotectant profile against fipronil (50 and 150μM) and fipronil sulfone (3 and 10μM) reaching control levels. Fipronil (100μM) and fipronil sulfone (3μM) treatments induced a 4.7- and 5-fold increases in lipid peroxides measured as malondialdehyde (MDA) and a 2.2- and 2.0-fold increases in the levels of nitric oxide (NO). These results suggest that oxidative stress observed may be one of the major mechanisms of fipronil-induced neurotoxicity and it may be attributed in part to fipronil disposition and metabolism. Our results led us postulate that metabolite fipronil sulfone might be responsible for the fipronil-induced toxicity rather than fipronil itself.

Plasma disposition and tissue depletion of chlortetracycline in the food producing animals, chickens for fattening.

Chickens were used to investigate plasma disposition of chlortetracycline after single IV (15 mg/kg) and multiple oral administration (60 mg/kg, 5 days) and residue depletion of chlortetracycline after multiple oral doses (60 mg/kg, 5 days). Plasma and tissue samples were analyzed by HPLC. Mean elimination half-lives in plasma were 7.96 and 13.15 h after IV and multiple oral administration. Maximum plasma concentration was 4.33 μg/ml and the interval from oral administration until maximal concentration was 1.79 h. Oral bioavailability was 17.76%. After multiple oral dose, mean kidney, liver and muscle tissue concentrations of chlortetracycline+4-epi-chlortetracycline of 835.3, 192.7, and 126.3 μg/kg, respectively, were measured 1 day after administration of the final dose of chlortetracycline. Chlortetracycline residues were detected in kidney and liver (205.4 and 81.7 μg/kg, respectively), but not in muscle, 3 days after the end of chlortetracycline treatment. The mean chlortetracycline+4-epi-chlortetracycline concentrations were below LOQ at 3 and 5 days after cessation of medication in muscle and liver, respectively. A withdrawal time of 3 days was necessary to ensure that the chlortetracycline residues were less than the maximal residue limits (MRLs) established by the European Union (100, 300, and 600 μg/kg in muscle, liver, and kidney, respectively).