Remi Bars - Academia.edu (original) (raw)
Papers by Remi Bars
Archives of Toxicology
With an increasing need to incorporate new approach methodologies (NAMs) in chemical risk assessm... more With an increasing need to incorporate new approach methodologies (NAMs) in chemical risk assessment and the concomitant need to phase out animal testing, the interpretation of in vitro assay readouts for quantitative hazard characterisation becomes more important. Physiologically based kinetic (PBK) models, which simulate the fate of chemicals in tissues of the body, play an essential role in extrapolating in vitro effect concentrations to in vivo bioequivalent exposures. As PBK-based testing approaches evolve, it will become essential to standardise PBK modelling approaches towards a consensus approach that can be used in quantitative in vitro-to-in vivo extrapolation (QIVIVE) studies for regulatory chemical risk assessment based on in vitro assays. Based on results of an ECETOC expert workshop, steps are recommended that can improve regulatory adoption: (1) define context and implementation, taking into consideration model complexity for building fit-for-purpose PBK models, (2) h...
ALTEX
Drug induced liver injury is a major problem for the pharmaceutical industry and health services.... more Drug induced liver injury is a major problem for the pharmaceutical industry and health services. Yet 30-40 % of human hepatotoxins go undetected during in vitro studies. Hence, more predictive in vitro liver models are a critical requirement for preclinical screening of compounds demonstrating hepatotoxic liability. 3D liver spheroids show promise as a novel model to investigate drug-induced liver injury with preliminary studies indicating the ability of spheroids to detect hepatotoxins as well as displaying an enhanced functional lifespan compared to 2D monocultures. The aim of this thesis was to develop an improved in vitro model to investigate druginduced liver injury. A viable C3A spheroid model with a lifespan of 32 days was successfully optimised. A characterisation of the spheroid model was performed, revealing direct cell-cell contacts, 3D morphology and cellular polarisation, hence recapitulating corresponding features of human liver tissue. Subsequently, liverspecific functions were investigated in the C3A spheroids and were found to display zonation, functional transporters, CYP enzyme activity, albumin production, urea synthesis and functional mitochondria. After validating the biology of the model, the ability of the spheroids to detect hepatotoxins was examined. The C3A spheroid model correctly identified 66.6 % of hepatotoxins to have a risk of liver injury, a higher predictive value than a 2D model. As hepatocytes only represent 60 % of the cells in the liver it was predicted that including non-parenchymal cells in the C3A spheroid model would cause increased sensitivity to hepatotoxins. Indeed when C3A spheroids were co-cultured with endothelial cells or immune cells they correctly predicted more compounds to have a risk of human hepatotoxicity, improving their predictivity to 91.6 % and 83.3 % respectively.
Biochemical Journal, 1992
The expression and induction of the cytochrome P450 2B1/2 isoenzyme is heterogeneous, exhibiting ... more The expression and induction of the cytochrome P450 2B1/2 isoenzyme is heterogeneous, exhibiting a regional pattern in the intact liver and a varied response to phenobarbital in isolated cultured hepatocytes. We report that P450 2B1/2 immunostaining of hepatocytes isolated from the perivenous liver region and cultured in the presence of phenobarbital is much stronger than that of cells identically treated but isolated from the periportal region. P450 2B1 mRNA, quantified by a sensitive and specific RNAase protection assay, is also preferentially induced in perivenous hepatocytes, demonstrating that the difference in induced expression is at the pretranslational level. Our results suggest that perivenous and periportal hepatocytes are differentially imprinted to retain regiospecific factors governing their inducibility after isolation.
Biochemical Journal, 1991
Rats received various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Aroclor 1254 (ARO), be... more Rats received various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Aroclor 1254 (ARO), beta-naphthoflavone (BNF) or phenobarbital (PB), and the hepatic expression of cytochromes P450IA1 and/or P450IIB1/IIB2 was analysed by immunohistochemistry and Western blotting. A clear heterogeneous acinar induction of IA1 was detected when a low dose of TCDD, ARO or BNF was administered. When a low dose of TCDD or ARO was administered, IA1 was found to be induced primarily in hepatocytes located in acinus zone 3, whereas when a low dose of BNF was administered, IA1 was found to be preferentially induced in hepatocytes located in acinus zone 1. A clear zonal induction of IIB1/IIB2 was also observed when a low dose of PB or ARO was administered. Both compounds induced IIB1/IIB2 preferentially in hepatocytes located in acinus zone 3. When rats were administered high doses of TCDD, ARO, BNF or PB there was no zonal pattern of induction of IA1 or IIB1/IIB2; instead, a pan-acinar induction of...
Biochemical Journal, 1991
The peroxisome proliferators are structurally diverse chemicals which induce hyperplasia, hypertr... more The peroxisome proliferators are structurally diverse chemicals which induce hyperplasia, hypertrophy and the proliferation of peroxisomes in the rodent liver. Cytochrome P450IVA1 and peroxisomal enzymes, such as acyl-CoA oxidase, are induced and are early markers of treatment with peroxisome proliferators. In this study, rats were dosed intraperitoneally with the potent peroxisome proliferator methylclofenapate and the hepatic induction response was studied. There was no significant change in the enzyme activities of laurate hydroxylase (cytochrome P450IVA1) or acyl-CoA oxidase in the first 8 h after treatment, but the activities had doubled at 24 h, suggesting that these enzymes are not involved in the mediation of early events in peroxisome proliferation. Hepatic cytochrome P450IVA1 mRNA was significantly increased at 6 and 8 h after treatment, rising to 15-fold above control values at 30 h. In contrast, acyl-CoA oxidase mRNA showed no significant change in the first 8 h, but inc...
Biochemical Journal, 1989
Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthof... more Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthoflavone, isosafrole or dexamethasone for 3 days, and the induction of several cytochrome P-450 isoenzymes was demonstrated by increased catalytic activity, by Western blotting and by immunocytochemistry. The profiles of isoenzymes induced in vitro were compared with those induced in liver microsomes of rats dosed with the same agents. Clofibric acid, an agent which has not been thoroughly investigated previously, was shown to induce both in vivo and in vitro several P-450 isoenzymes normally inducible by phenobarbitone (PB1a, PB3a and PB3b) or steroids (PB2c). Immunocytochemical studies demonstrated that the inducible isoenzymes of cytochrome P-450 are not distributed evenly throughout the hepatocyte population, and increasing concentrations of phenobarbitone or beta-naphthoflavone in the medium results in an increasing proportion of ‘induced’ cells. However, whereas maximal concentration...
Regulatory Toxicology and Pharmacology, 2009
Genetically modified crops convey many benefits to world population. However, a rigorous safety a... more Genetically modified crops convey many benefits to world population. However, a rigorous safety assessment procedure, including an evaluation of the allergenic potential, is fundamental before their release into the food chain. As an integral part of the safety assessment process, regulatory authorities worldwide strongly recommend the use of tests that can predict the allergenic potential of the novel proteins. All guidance documents are based on an array of tests that have been proposed in 2003 by the Codex Alimentarius. Although the animal model is not a requirement of the Codex Alimentarius weight of evidence approach, allergenic hazard of novel proteins could only be evaluated by an in vivo model that can potentially identify and distinguish commonly allergenic proteins from rarely allergenic proteins. Therefore, food allergy experts encourage its development. During the 2007 International Life Science Institute (ILSI) workshop (Nice, France), worldwide experts shared their latest research results on rodent models to evaluate the allergenic potential of proteins and foods. This review presents the most promising rodent models for assessing food protein allergenicity that were evaluated during this ILSI workshop.
Testicular Tangrams, 2002
Environmental chemicals which mimic or antagonize the actions of steroid hormones have the potent... more Environmental chemicals which mimic or antagonize the actions of steroid hormones have the potential to disrupt endocrine function and could potentially pose a threat to human health [1–3]. Different studies have documented the ability of these chemicals to interfere with male gonadal formation and function in experimental models [4, 5]. As steroid hormones play a critical role in the early development of the genital tract [6], it has been hypothesized that some synthetic chemicals in the environment could affect adult male reproductive organs by stimulating or inhibiting receptor mediated developmental events following an in utero exposure [5]. Several reports in the literature have indicated that in utero exposure to exogenous anti-androgenic compounds can induce a wide range of abnormalities of the reproductive system, including small testes, cryptorchidism and hypospadia [5–8]. These compounds have been reported to interact with the androgen receptor [7, 8]. For example, the drug flutamide and its active metabolite hydroxyflutamide are non-steroidal synthetic chemicals able to inhibit the action of androgens at the receptor level [9]. In utero exposure to flutamide has been shown to induce major alterations in the accessory sex glands and in testis development in male rat offspring [5–8]. Exposure to this antiandrogen resulted in a decrease in the gonad weight with marked testicular morphological alterations including a reduction in average diameter of the seminiferous tubules associated with moderate to severe hypospermatogenesis with an interruption of germ cell maturation [8]. The cellular and molecular mechanisms underlying the arrest of the spermatogenetic process resulting in the loss of the mature germ cells remain to be investigated.
Regulatory Toxicology and Pharmacology, 2014
Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mo... more Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mold, botrytis). During the general toxicity evaluation of fluopyram in rodents, the liver was identified as a target organ (hepatomegaly and liver hypertrophy were observed in all studies). At the end of the guideline carcinogenicity study, an increased incidence of hepatocellular adenomas and carcinomas was observed in female Wistar rats following exposure to the highest fluopyram dose evaluated (1500 ppm). Short-term mechanistic studies (3, 7 or 28 days of exposure) were conducted in the female rat to identify the initial key events responsible for the tumor formation and to establish thresholds for each of the early hepatic changes. Increased expression of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) inducible genes was recorded after each exposure period. Further confirmation of CAR/PXR activation was provided by increased activity of specific Phase I enzymes (PROD/BROD respectively). Increased hepatocellular proliferation (measured by Ki67) was observed after each exposure period with the greatest proliferative response occurring after 3 days of treatment. In these studies, dose responses and clear thresholds were established for gene expression, enzyme activity and cell proliferation. Furthermore, these early hepatic changes were shown to be reversible following compound withdrawal. Other modes of action for liver tumor formation such as DNA damage, cytotoxicity and peroxisome proliferation were excluded during the investigations. In conclusion, fluopyram is a threshold carcinogen and the resultant hepatocellular carcinomas in the female rat are due to hepatocellular proliferation mediated by CAR/PXR activation.
Toxicology in Vitro, 2003
The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte micro... more The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2-and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. l-NAME (1 mm) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of l-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of l-NAME on nitric oxide synthesis inhibition. The present work also shows that l-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes.
Toxicology in Vitro, 2001
Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Follow... more Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Following isolation, hepatocytes produce large amounts of nitric oxide (NO), which is known to have both pro-and anti-apoptotic effects in hepatocytes in vivo and in vitro. Previous work has not determined the effect of these increased levels of NO on the response of hepatocytes to apoptotic stimuli. Here we report that levels of nitrites are elevated in hepatocyte monolayers from 24 h onwards. Addition of the inducible nitric oxide synthase (iNOS) inhibitor, No-nitro-l-arginine methyl ester (L-NAME), to the medium inhibited this increase in nitrites. These results indicate that the increase in nitrite is most likely due to the formation of NO. Elevated nitrite levels had no effect either on basal levels of apoptosis or on ATP and GSH. Apoptosis was induced by transforming growth factor b-1 (TGFb-1) or glycochenodeoxycholate (GCDC). Both compounds caused moderate hepatocyte apoptosis; however, addition of L-NAME prior to exposure significantly increased the level of apoptosis observed with the two compounds. Both TGFb-1 and GCDC had no effect on hepatocyte ATP or GSH levels; however, as a consequence of secondary necrosis, TGFb-1 exposure significantly increased levels of lactate dehydrogenase (LDH) leakage. These findings indicate that the increased levels of NO associated with the culture of hepatocytes have an inhibitory effect on compound-induced apoptosis in the cells.
Toxicology in Vitro, 2002
We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbitu... more We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or threedimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)-and UDP-glucuronosyl transferase (UGT)-dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT-and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.
Toxicological Sciences, 2011
Reproductive Toxicology, 2013
Reproductive Toxicology, 2004
Three chemicals with known endocrine activities have been evaluated in the rat Hershberger assay ... more Three chemicals with known endocrine activities have been evaluated in the rat Hershberger assay for phase-2 of the international validation exercise within the Organization for Economic Cooperation and Development (OECD). The chemicals studied included the antiandrogens finasteride (FIN) and procymidone (PRO) and the androgen agonist 17alpha-methyltestosterone (MT). Castration of sexually immature Sprague-Dawley rats was performed between post-natal days 42 and 46 whilst dosing of the chemical over 10 days was performed between post-natal days 53 and 67. Rats were co-treated with testosterone propionate (TP) for the antiandrogenic activity evaluation. The endpoints examined for evaluation of the androgenic/antiandrogenic activity were changes in sex accessory tissue (SAT) weights supplemented with measurement of testosterone and luteinizing hormone (LH) levels at sacrifice. Changes in liver, adrenal, kidney and body weights were also monitored for general toxicity assessment. Statistically significant changes in the SAT weights were detected with the three chemicals tested. Hence, the rat Hershberger assay as defined by the OECD was demonstrated sensitive enough for the detection of the endocrine disrupting activity of the three reference chemicals evaluated.
Regulatory Toxicology and Pharmacology, 2011
The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocide... more The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocides (Directive 98/8/EC), as well as the regulation concerning chemicals (Regulation (EC) No. 1907/2006 'REACH') only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or non-target species. However, there is currently no agreed guidance on how to identify and evaluate endocrine activity and disruption. Consequently, an ECETOC task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. Specific scientific criteria for the determination of endocrine disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies are proposed. These criteria combine the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. The criteria developed are presented in the form of flow charts for assessing relevant effects for both humans and wildlife species. In addition, since not all chemicals with endocrine disrupting properties are of equal hazard, assessment of potency is also proposed to discriminate chemicals of high concern from those of lower concern. The guidance presented in this paper includes refinements made to an initial proposal following discussion of the criteria at a workshop of invited regulatory, academic and industry scientists.
Regulatory Toxicology and Pharmacology, 2013
Archives of Toxicology
With an increasing need to incorporate new approach methodologies (NAMs) in chemical risk assessm... more With an increasing need to incorporate new approach methodologies (NAMs) in chemical risk assessment and the concomitant need to phase out animal testing, the interpretation of in vitro assay readouts for quantitative hazard characterisation becomes more important. Physiologically based kinetic (PBK) models, which simulate the fate of chemicals in tissues of the body, play an essential role in extrapolating in vitro effect concentrations to in vivo bioequivalent exposures. As PBK-based testing approaches evolve, it will become essential to standardise PBK modelling approaches towards a consensus approach that can be used in quantitative in vitro-to-in vivo extrapolation (QIVIVE) studies for regulatory chemical risk assessment based on in vitro assays. Based on results of an ECETOC expert workshop, steps are recommended that can improve regulatory adoption: (1) define context and implementation, taking into consideration model complexity for building fit-for-purpose PBK models, (2) h...
ALTEX
Drug induced liver injury is a major problem for the pharmaceutical industry and health services.... more Drug induced liver injury is a major problem for the pharmaceutical industry and health services. Yet 30-40 % of human hepatotoxins go undetected during in vitro studies. Hence, more predictive in vitro liver models are a critical requirement for preclinical screening of compounds demonstrating hepatotoxic liability. 3D liver spheroids show promise as a novel model to investigate drug-induced liver injury with preliminary studies indicating the ability of spheroids to detect hepatotoxins as well as displaying an enhanced functional lifespan compared to 2D monocultures. The aim of this thesis was to develop an improved in vitro model to investigate druginduced liver injury. A viable C3A spheroid model with a lifespan of 32 days was successfully optimised. A characterisation of the spheroid model was performed, revealing direct cell-cell contacts, 3D morphology and cellular polarisation, hence recapitulating corresponding features of human liver tissue. Subsequently, liverspecific functions were investigated in the C3A spheroids and were found to display zonation, functional transporters, CYP enzyme activity, albumin production, urea synthesis and functional mitochondria. After validating the biology of the model, the ability of the spheroids to detect hepatotoxins was examined. The C3A spheroid model correctly identified 66.6 % of hepatotoxins to have a risk of liver injury, a higher predictive value than a 2D model. As hepatocytes only represent 60 % of the cells in the liver it was predicted that including non-parenchymal cells in the C3A spheroid model would cause increased sensitivity to hepatotoxins. Indeed when C3A spheroids were co-cultured with endothelial cells or immune cells they correctly predicted more compounds to have a risk of human hepatotoxicity, improving their predictivity to 91.6 % and 83.3 % respectively.
Biochemical Journal, 1992
The expression and induction of the cytochrome P450 2B1/2 isoenzyme is heterogeneous, exhibiting ... more The expression and induction of the cytochrome P450 2B1/2 isoenzyme is heterogeneous, exhibiting a regional pattern in the intact liver and a varied response to phenobarbital in isolated cultured hepatocytes. We report that P450 2B1/2 immunostaining of hepatocytes isolated from the perivenous liver region and cultured in the presence of phenobarbital is much stronger than that of cells identically treated but isolated from the periportal region. P450 2B1 mRNA, quantified by a sensitive and specific RNAase protection assay, is also preferentially induced in perivenous hepatocytes, demonstrating that the difference in induced expression is at the pretranslational level. Our results suggest that perivenous and periportal hepatocytes are differentially imprinted to retain regiospecific factors governing their inducibility after isolation.
Biochemical Journal, 1991
Rats received various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Aroclor 1254 (ARO), be... more Rats received various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Aroclor 1254 (ARO), beta-naphthoflavone (BNF) or phenobarbital (PB), and the hepatic expression of cytochromes P450IA1 and/or P450IIB1/IIB2 was analysed by immunohistochemistry and Western blotting. A clear heterogeneous acinar induction of IA1 was detected when a low dose of TCDD, ARO or BNF was administered. When a low dose of TCDD or ARO was administered, IA1 was found to be induced primarily in hepatocytes located in acinus zone 3, whereas when a low dose of BNF was administered, IA1 was found to be preferentially induced in hepatocytes located in acinus zone 1. A clear zonal induction of IIB1/IIB2 was also observed when a low dose of PB or ARO was administered. Both compounds induced IIB1/IIB2 preferentially in hepatocytes located in acinus zone 3. When rats were administered high doses of TCDD, ARO, BNF or PB there was no zonal pattern of induction of IA1 or IIB1/IIB2; instead, a pan-acinar induction of...
Biochemical Journal, 1991
The peroxisome proliferators are structurally diverse chemicals which induce hyperplasia, hypertr... more The peroxisome proliferators are structurally diverse chemicals which induce hyperplasia, hypertrophy and the proliferation of peroxisomes in the rodent liver. Cytochrome P450IVA1 and peroxisomal enzymes, such as acyl-CoA oxidase, are induced and are early markers of treatment with peroxisome proliferators. In this study, rats were dosed intraperitoneally with the potent peroxisome proliferator methylclofenapate and the hepatic induction response was studied. There was no significant change in the enzyme activities of laurate hydroxylase (cytochrome P450IVA1) or acyl-CoA oxidase in the first 8 h after treatment, but the activities had doubled at 24 h, suggesting that these enzymes are not involved in the mediation of early events in peroxisome proliferation. Hepatic cytochrome P450IVA1 mRNA was significantly increased at 6 and 8 h after treatment, rising to 15-fold above control values at 30 h. In contrast, acyl-CoA oxidase mRNA showed no significant change in the first 8 h, but inc...
Biochemical Journal, 1989
Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthof... more Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthoflavone, isosafrole or dexamethasone for 3 days, and the induction of several cytochrome P-450 isoenzymes was demonstrated by increased catalytic activity, by Western blotting and by immunocytochemistry. The profiles of isoenzymes induced in vitro were compared with those induced in liver microsomes of rats dosed with the same agents. Clofibric acid, an agent which has not been thoroughly investigated previously, was shown to induce both in vivo and in vitro several P-450 isoenzymes normally inducible by phenobarbitone (PB1a, PB3a and PB3b) or steroids (PB2c). Immunocytochemical studies demonstrated that the inducible isoenzymes of cytochrome P-450 are not distributed evenly throughout the hepatocyte population, and increasing concentrations of phenobarbitone or beta-naphthoflavone in the medium results in an increasing proportion of ‘induced’ cells. However, whereas maximal concentration...
Regulatory Toxicology and Pharmacology, 2009
Genetically modified crops convey many benefits to world population. However, a rigorous safety a... more Genetically modified crops convey many benefits to world population. However, a rigorous safety assessment procedure, including an evaluation of the allergenic potential, is fundamental before their release into the food chain. As an integral part of the safety assessment process, regulatory authorities worldwide strongly recommend the use of tests that can predict the allergenic potential of the novel proteins. All guidance documents are based on an array of tests that have been proposed in 2003 by the Codex Alimentarius. Although the animal model is not a requirement of the Codex Alimentarius weight of evidence approach, allergenic hazard of novel proteins could only be evaluated by an in vivo model that can potentially identify and distinguish commonly allergenic proteins from rarely allergenic proteins. Therefore, food allergy experts encourage its development. During the 2007 International Life Science Institute (ILSI) workshop (Nice, France), worldwide experts shared their latest research results on rodent models to evaluate the allergenic potential of proteins and foods. This review presents the most promising rodent models for assessing food protein allergenicity that were evaluated during this ILSI workshop.
Testicular Tangrams, 2002
Environmental chemicals which mimic or antagonize the actions of steroid hormones have the potent... more Environmental chemicals which mimic or antagonize the actions of steroid hormones have the potential to disrupt endocrine function and could potentially pose a threat to human health [1–3]. Different studies have documented the ability of these chemicals to interfere with male gonadal formation and function in experimental models [4, 5]. As steroid hormones play a critical role in the early development of the genital tract [6], it has been hypothesized that some synthetic chemicals in the environment could affect adult male reproductive organs by stimulating or inhibiting receptor mediated developmental events following an in utero exposure [5]. Several reports in the literature have indicated that in utero exposure to exogenous anti-androgenic compounds can induce a wide range of abnormalities of the reproductive system, including small testes, cryptorchidism and hypospadia [5–8]. These compounds have been reported to interact with the androgen receptor [7, 8]. For example, the drug flutamide and its active metabolite hydroxyflutamide are non-steroidal synthetic chemicals able to inhibit the action of androgens at the receptor level [9]. In utero exposure to flutamide has been shown to induce major alterations in the accessory sex glands and in testis development in male rat offspring [5–8]. Exposure to this antiandrogen resulted in a decrease in the gonad weight with marked testicular morphological alterations including a reduction in average diameter of the seminiferous tubules associated with moderate to severe hypospermatogenesis with an interruption of germ cell maturation [8]. The cellular and molecular mechanisms underlying the arrest of the spermatogenetic process resulting in the loss of the mature germ cells remain to be investigated.
Regulatory Toxicology and Pharmacology, 2014
Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mo... more Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mold, botrytis). During the general toxicity evaluation of fluopyram in rodents, the liver was identified as a target organ (hepatomegaly and liver hypertrophy were observed in all studies). At the end of the guideline carcinogenicity study, an increased incidence of hepatocellular adenomas and carcinomas was observed in female Wistar rats following exposure to the highest fluopyram dose evaluated (1500 ppm). Short-term mechanistic studies (3, 7 or 28 days of exposure) were conducted in the female rat to identify the initial key events responsible for the tumor formation and to establish thresholds for each of the early hepatic changes. Increased expression of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) inducible genes was recorded after each exposure period. Further confirmation of CAR/PXR activation was provided by increased activity of specific Phase I enzymes (PROD/BROD respectively). Increased hepatocellular proliferation (measured by Ki67) was observed after each exposure period with the greatest proliferative response occurring after 3 days of treatment. In these studies, dose responses and clear thresholds were established for gene expression, enzyme activity and cell proliferation. Furthermore, these early hepatic changes were shown to be reversible following compound withdrawal. Other modes of action for liver tumor formation such as DNA damage, cytotoxicity and peroxisome proliferation were excluded during the investigations. In conclusion, fluopyram is a threshold carcinogen and the resultant hepatocellular carcinomas in the female rat are due to hepatocellular proliferation mediated by CAR/PXR activation.
Toxicology in Vitro, 2003
The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte micro... more The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2-and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. l-NAME (1 mm) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of l-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of l-NAME on nitric oxide synthesis inhibition. The present work also shows that l-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes.
Toxicology in Vitro, 2001
Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Follow... more Primary cultures of hepatocytes are a widely used in vitro model for biochemical research. Following isolation, hepatocytes produce large amounts of nitric oxide (NO), which is known to have both pro-and anti-apoptotic effects in hepatocytes in vivo and in vitro. Previous work has not determined the effect of these increased levels of NO on the response of hepatocytes to apoptotic stimuli. Here we report that levels of nitrites are elevated in hepatocyte monolayers from 24 h onwards. Addition of the inducible nitric oxide synthase (iNOS) inhibitor, No-nitro-l-arginine methyl ester (L-NAME), to the medium inhibited this increase in nitrites. These results indicate that the increase in nitrite is most likely due to the formation of NO. Elevated nitrite levels had no effect either on basal levels of apoptosis or on ATP and GSH. Apoptosis was induced by transforming growth factor b-1 (TGFb-1) or glycochenodeoxycholate (GCDC). Both compounds caused moderate hepatocyte apoptosis; however, addition of L-NAME prior to exposure significantly increased the level of apoptosis observed with the two compounds. Both TGFb-1 and GCDC had no effect on hepatocyte ATP or GSH levels; however, as a consequence of secondary necrosis, TGFb-1 exposure significantly increased levels of lactate dehydrogenase (LDH) leakage. These findings indicate that the increased levels of NO associated with the culture of hepatocytes have an inhibitory effect on compound-induced apoptosis in the cells.
Toxicology in Vitro, 2002
We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbitu... more We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or threedimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)-and UDP-glucuronosyl transferase (UGT)-dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT-and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.
Toxicological Sciences, 2011
Reproductive Toxicology, 2013
Reproductive Toxicology, 2004
Three chemicals with known endocrine activities have been evaluated in the rat Hershberger assay ... more Three chemicals with known endocrine activities have been evaluated in the rat Hershberger assay for phase-2 of the international validation exercise within the Organization for Economic Cooperation and Development (OECD). The chemicals studied included the antiandrogens finasteride (FIN) and procymidone (PRO) and the androgen agonist 17alpha-methyltestosterone (MT). Castration of sexually immature Sprague-Dawley rats was performed between post-natal days 42 and 46 whilst dosing of the chemical over 10 days was performed between post-natal days 53 and 67. Rats were co-treated with testosterone propionate (TP) for the antiandrogenic activity evaluation. The endpoints examined for evaluation of the androgenic/antiandrogenic activity were changes in sex accessory tissue (SAT) weights supplemented with measurement of testosterone and luteinizing hormone (LH) levels at sacrifice. Changes in liver, adrenal, kidney and body weights were also monitored for general toxicity assessment. Statistically significant changes in the SAT weights were detected with the three chemicals tested. Hence, the rat Hershberger assay as defined by the OECD was demonstrated sensitive enough for the detection of the endocrine disrupting activity of the three reference chemicals evaluated.
Regulatory Toxicology and Pharmacology, 2011
The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocide... more The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocides (Directive 98/8/EC), as well as the regulation concerning chemicals (Regulation (EC) No. 1907/2006 'REACH') only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or non-target species. However, there is currently no agreed guidance on how to identify and evaluate endocrine activity and disruption. Consequently, an ECETOC task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. Specific scientific criteria for the determination of endocrine disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies are proposed. These criteria combine the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. The criteria developed are presented in the form of flow charts for assessing relevant effects for both humans and wildlife species. In addition, since not all chemicals with endocrine disrupting properties are of equal hazard, assessment of potency is also proposed to discriminate chemicals of high concern from those of lower concern. The guidance presented in this paper includes refinements made to an initial proposal following discussion of the criteria at a workshop of invited regulatory, academic and industry scientists.
Regulatory Toxicology and Pharmacology, 2013