Assessment of deoxynivalenol (DON) adsorbents and characterisation of their efficacy using complementary in vitro tests.

Deoxynivalenol (DON) is a prevalent and resistant mycotoxin found in cereals and related products. Adsorbents appear to provide an opportunity to decrease DON absorption in animals but, due to their specificity, it is very difficult to evaluate their actual efficacy. It is pointless to extrapolate results obtained with one mycotoxin to another and even to extrapolate results obtained in vitro in buffer to an in vivo situation. We carried out experiments to characterize the properties of potential DON adsorbents. Initial tests in buffer pH 7 allowed us to focus on six adsorbents: activated charcoal, cholestyramin, Saccharomyces cerevisiae mannans, algal beta-glycan, fungal beta-glycan and leguminous plant. The use of equilibrium sorption models suggested a non-saturated phenomenon and involved variable mechanisms according to the specific material. Subsequent tests with a Caco-2 cell model showed a high reduction in DON cytotoxicity on proliferative intestinal cells and DON absorption by differentiated intestinal cells when adsorbent was added (except for cholestyramin). Otherwise, values were not always in accordance with those obtained in buffer. Our work allowed us to identify five potential DON adsorbents and to propose a complementary in vitro test allowing improved determination of adsorbent properties.

Fusariotoxin transfer in animal.

Mycotoxin fusariotoxins, essentially represented by trichothecenes, zearalenone and fumonisins, are widely scattered in cereals and their products. Human and animals are particularly concerned by toxicity consecutive to oral chronic exposure. Human exposure can be direct via cereals or indirect via products of animals having eaten contaminated feed. As this alimentary risk is considered as a major problem in public health, it is thus of great importance to determine bioavailability, metabolic pathways and distribution of these mycotoxins in animal and human organism. Most studies indicate that fusariotoxins can be rapidly absorbed in the small intestine but the mechanisms involved remain unclear. Except NIV, fusariotoxins can be partly metabolised into more hydrophilic molecules in digestive tract or liver. Fumonisins present different behaviour as they seem very few and slowly absorbed and metabolised. The main part of absorbed fusariotoxins shows a rapid elimination within 24h after ingestion, followed by a slower excretion of small amounts. However, traces of fusariotoxins or their derivates can be found in animal products. This manuscript, reviewing literature published on fusariotoxin transfer, highlights that too little data are available to correctly appreciate fusariotoxin transfer in organism. Further studies focusing on mechanisms involved in the transfer are needed before clarifying risk assessment for human health.

Diazinon cytotoxicity and transfer in Caco-2 cells: Effect of long-term exposure to the pesticide.

The purpose of this work was to investigate the effect of prolonged exposure to diazinon (widely used organophosphorus pesticide) on the intestinal cell-line Caco-2. Cytotoxicity of the pesticide (50µM-6mM) significantly decreased in long-term exposed (20µM, 2 months) cells, compared to untreated control cells. In long-term exposed cells, the resistance to diazinon cytotoxicity was reversed in the presence of PSC-833, a P-glycoprotein (P-gp) inhibitor, but not in the presence of MK 571, a Multidrug Resistance Protein (MRP) inhibitor. Cell exposure to 25µM diazinon showed a secretory-directed transport of the molecule, which increased in long-term exposed cells. This efflux decreased significantly, for both long-term and non-exposed cells, in the presence of verapamil and PSC-833, but not MK 571. Furthermore, the total amount of P-gp increased in long-term exposed cells. These results suggest that ABC transporter P-gp is involved in the intestinal transfer of diazinon, and that repeated exposure to low doses of diazinon could strengthen the activity of ABC transporters in intestinal cells, thus increasing cell resistance to pesticide cytotoxicity.

Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha.

The effects of metal mixture (Cd+Cu) versus single-metal exposure on total MT response and bioaccumulation were investigated in the freshwater bivalve Dreissena polymorpha. A two-month exposure period, including two levels of contamination, was chosen for each of the two metals: 5, 10 microg/L for Cu, and 2, 20 microg/L for Cd, with mixtures of, respectively, 5 microg/L Cu+2 microg/L Cd, 5 microg/L Cu+20 microg/L Cd, 10 microg/L Cu+2 microg/L Cd, and 10 microg/L Cu+20 microg/L Cd. Total MT contents were assessed by an Ag-saturation method, and metals contents were determined by atomic absorption spectrometry. Results at the whole-organism level showed a significant and early increase of total MT biosynthesis after exposure to Cd. This increase was significantly correlated with Cd bioaccumulation. By contrast, Cu did not modify total MT response, and mussels limited Cu bioaccumulation. The mixture either did not influence or only weakly influenced metal accumulation and MT response to Cu and Cd after long-term exposure. Our results suggest that the form of MT existing in D. polymorpha was not Cu-inducible. This could limit the use of MT in D. polymorpha as a biomarker of heavy metal pollution in freshwater ecosystems.

Implication of distinct proteins in cadmium uptake and transport by intestinal cells HT-29.

The mechanisms of intestinal absorption have not been clearly elucidated for cadmium, a toxic metal. In this work, we show the implication of distinct proteins in cadmium transport, and the transport step where these proteins are involved. We first validated the HT-29 model by evaluating nontoxic doses of cadmium (ranging from 1 to 20 micromol/L), and by quantifying metal uptake and transepithelial transport. The time-course of 1 micromol/L cadmium uptake at pH 7.5 showed three steps: a rapid one during the first 4 min, probably due to cadmium binding to the membrane; a slower one, characterized by Km of 1.65+/-0.54 micromol/L and Vmax of 3.9+/-0.3 micromol/min per mg protein; and a third, corresponding to slow accumulation that was not equilibrated even after 48 h of cadmium exposure. Intracellular metallothionein content following 1 or 5 micromol/L cadmium exposure showed a significant increase after 6 h of exposure, and was not equilibrated even after 72 h, allowing cadmium accumulation. After 24 h of exposure, metallothionein content was 5-fold, 14-fold, 26-fold, and 50-fold, respectively, for cells grown in the presence of 1, 5, 10, and 20 micromol/L cadmium, compared to control cells. The second step of uptake, characterized by carrier-mediated transport, was markedly increased at pH 5.5, compared to pH 7.5, and strongly inhibited by the metabolic inhibitor dinitrophenol. Moreover Nramp2 transporter cDNA was present in HT-29 cells. These data suggest the involvement of a proton-coupled transporter, which may be the divalent cation transporter Nramp2 (natural resistance-associated macrophage protein 2). Cadmium uptake was also inhibited by copper, zinc, and para-chloromercuribenzenesulfonate (pCMBS), but not by verapamil or ouabain. Taken together, our results indicate that cadmium could enter HT-29 cell by Nramp2 proton-coupled active transport and by diffusion, and accumulates in the cell as long as it binds to metallothionein. Cadmium toxicity could depend partly on the activity of Nramp2, and partly on metallothionein content.

Cadmium uptake and transepithelial transport in control and long-term exposed Caco-2 cells: the role of metallothionein.

Exposure of humans to cadmium, a common environmental pollutant, is mainly through food intake. However, the mechanisms of intestinal absorption have not been clearly elucidated for this toxic metal ion. In order to investigate the effects of long-term exposure to this metal and the role of metallothioneins in cadmium absorption, we used human-derived Caco-2 cells cultured on porous membrane filters. We first validated this model by quantifying metal uptake and transepithelial transport on control cells and cells adapted to grow for 2 to 5 weeks in the presence of low doses of cadmium in the culture medium. The nontoxic doses of cadmium (0.1, 1.0, and 5 microM), in which Caco-2 cells could be cultured for many passages without deleterious effects, were determined by evaluating transepithelial resistance of the cells and lactate dehydrogenase leakage. After 24 h of 1 microM Cd exposure, intracellular cadmium levels were 3- and 6-fold higher for cells exposed for extended periods to 1 and 5 microM cadmium, respectively, compared to control cells. In control and long-term exposed cells, this accumulation was inhibited by zinc, copper, and pCMBS, but not by verapamil or ouabain. Intracellular metallothionein content was increased 1.5-, 5-, and 12-fold for the cells grown in the presence of 0.1, 1.0, and 5 microM cadmium, respectively, in the culture medium. The amount of metallothionein synthesized and released by the cells was highly correlated with cadmium accumulation and transport. Our results suggest that Caco-2 cell monolayers are a good predictive model for the study of cadmium intestinal absorption following exposure to repeated low doses of cadmium, and confirm the essential role of metallothionein in the regulation of cadmium intestinal absorption.

Covalent binding of carbamazepine reactive metabolites to P450 isoforms present in the skin.

Carbamazepine is an anticonvulsant associated with a high risk for severe cutaneous reactions. Upon metabolism by cytochrome P450, carbamazepine may produce reactive metabolites. We evaluated in vitro the covalent binding of carbamazepine reactive metabolites on human P450s and then the presence of these P450s in human epidermis. Carbamazepine reactive metabolites covalent binding to human liver microsomes involved P450 subfamilies 1A, 2C and 3A. Specific covalent binding to yeasts expressing different P450s showed that carbamazepine reactive metabolites bound specifically to P450 1A2 and 3A4. We confirmed the constitutive presence of P450 3A in human epidermis and after induction with coaltar of P450 1A. Consequently, the production in epidermis of carbamazepine reactive metabolites is theoretically possible with formation of P450 adduct metabolites.

Immunotoxicology of the liver: adverse reactions to drugs.

Liver is a frequent target for drug-induced hepatitis. They can be classified in two categories: the hepatitis in which the drug or a metabolite reach a vital target in the cell and the hepatitis in which the drug triggers an adverse immune response directed against the liver. We will discuss essentially this second kind of disease. They have key clinical features such as the low frequency, the dose independence, the delay between the beginning of drug intake and the triggering of the disease, the shortening of the delay upon rechallenge and very often the presence of autoantibodies in the serum of the patients. Such signs were found in hepatitis triggered by drugs such as halothane, tienilic acid, dihydralazine, anticonvulsants. They will be taken as examples to show the recent progress in the understanding of the mechanisms leading to the disease. It has been postulated that the drug is metabolised into a reactive metabolite binding to the enzyme which generated it; therefore the neoantigen might trigger an immune response characterised by the production of antibodies recognising the native and or the modified protein. Most of these steps were proven in the cases of halothane, tienilic acid and dihydralazine. Several points seem important in the development of the disease; the equilibrium between toxication and detoxication pathways, the nature and amount of neoantigen, the individual immune response. However, many points remain unclear: for instance, the reason for the very low frequency of this kind of disease; the precise mechanism of the adverse immune response; the risk factors for developing such adverse reactions. Efforts should be made to better understand the mechanisms of this kind of disease: for instance, an animal model, tests to identify drugs at risk for such reactions, the role of these drugs in the processing of P450s and the processing of the neoantigens for their presentation to the immune system.

Contribution of human cytochrome P450 to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast.

The metabolism of benzo[a]pyrene (B[a]P) and its proximate mutagen B[a]P-7,8-dihydrodiol (7,8-diol) was investigated in the presence of human microsomal epoxide hydrolase and P450 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 expressed in the yeast Saccharomyces cerevisiae. P450 1A1 had the highest turnover numbers for the formation of all B[a]P metabolites, including phenols and dihydrodiols. P450 1A2, 2C8, 2C9, 2C18, 2C19 and 3A4, which are well represented in the liver, gave rise to the formation of appreciable amounts of 3-hydroxy-B[a]P and of some dihydrodiols from B[a]P. When 7,8-diol was used as substrate, P450 1A1 also exhibited the highest turnover numbers for the formation of tetrols, the hydrolysis products of the diolepoxides, whereas P450 1A2, 2C8, 2C19 and 3A4 showed moderate activities. In order to test the validity of the yeast system, the contribution of each P450 isoform to B[a]P and 7,8-diol metabolism was evaluated as the product of the turnover numbers of recombinant P450s by specific contents of each P450 in human liver microsomes. Calculated formation rates for each B[a]P and 7,8-diol metabolite globally matched experimental values. There is evidence that P450 3A4 and 2C9 play a major role in the formation of 3-hydroxy-B[a]P from B[a]P. Accumulation of the proximate mutagen 7,8-diol was predicted to be mainly driven by P450 1A2, 2C9 and 2C19, while formation of the genotoxic diolepoxides from 7,8-diol appeared to be dependent on P450 1A2 and 3A4 in the liver.

Tienilic acid-induced autoimmune hepatitis: anti-liver and-kidney microsomal type 2 autoantibodies recognize a three-site conformational epitope on cytochrome P4502C9.

Tienilic acid-induced hepatitis is characterized by the presence of anti-liver and -kidney microsomal (anti-LKM2) autoantibodies in patient sera. Cytochrome P4502C9(CYP2C9), involved in the metabolism of tienilic acid, was shown to be a target for tienilic acid-reactive metabolites and for autoantibodies. To further investigate the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, the specificity of anti-LKM2 autoantibodies toward CYP2C9 was first determined, and the antigenic sites on CYP2C9 were localized. By constructing several deletion mutants derived from CYP2C9 cDNA and by probing the corresponding proteins with different anti-LKM2 sera, we defined three regions (amino acids 314-322, 345-356, and 439-455); they interacted to form a major conformational autoantibody binding site. This binding site was immunoreactive with 100% of sera and allowed removal of the entire reactivity of the sera tested by immunoblotting. Epitope mapping studies have been performed for CYP2D6, CYP17, CYP21A2, and, recently, CYP3A. Those data were compared with the results obtained in the current study with CYP2C9 in an attempt to elucidate one of the mechanisms by which CYP becomes immunogenic.

Interindividual variability in catalytic activity and immunoreactivity of three major human liver cytochrome P450 isozymes.

OBJECTIVE: Interindividual variations in immunoreactivity and function of three major human drug metabolising P450 monooxygenases has been investigated in liver microsomes from 42 Caucasians (kidney donors or liver biopsies). METHODS: Diclofenac 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation, measured by HPLC in incubates, were used as probes to determine CYP2C9, CYP2D6 and CYP3A4 function kinetics, respectively. Immunoquantification of the three isoforms was achieved by Western blotting, using rabbit polyclonal antibodies raised against human CYP2C9 and human CYP3A4, and mouse monoclonal antibody raised against human CYP2D6. RESULTS: Diclofenac 4'-hydroxylation exhibited Michaelis-Menten kinetics with kM = 3.4 mumol.l-1 and Vmax = 45 nmole.mg-1 P.h-1. Relative immunoreactivity of CYP2C9 was correlated with Vmax and CL(int). Dextromethorphan O-demethylation in EM (extensive metabolisers) liver microsomes also showed Michaelis-Menten kinetics, with kM = 4.4 mumol.l-1 and Vmax = 5.0 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP2D6 was correlated with Vmax and CL(int). Midazolam 1'-hydroxylation also exhibited Michaelis-Menten kinetics with kM = 3.3 mumol.l-1 and Vmax = 35 nmol.mg-1 P.h-1. Relative immunoreactivity of CYP3A4 was correlated with Vmax and CL(int). Immunoreactivity and function were correlated for each isozyme, but there was no cross correlation between isozymes. CONCLUSION: The velocity of metabolite formation (Vmax) by the three major human drug metabolising P450 monoxygenases is correlated with their immunoreactivity in liver microsomes. Interindividual variation was much larger for Vmax than kM. Interindividual variability was more pronounced for CYP2D6, probably due to the presence of several different functional alleles in the population of extensive metabolisers.

Anti-cytochrome P450 autoantibodies in drug-induced disease.

Drugs may induce hepatitis through immune mechanisms. In this review we have used the examples of 2 drugs to elucidate the first steps leading to the triggering of such disease, namely tienilic acid (TA) and dihydralazine (DH). These drugs are transformed into reactive metabolite(s) by cytochrome P450 (2C9 for TA and 1A2 for DH) (step 1). The reactive metabolites produced are very short-lived and bind directly to the enzymes which generated them (step 2). A neoantigen is thus formed which triggers an immune response (step 3), characterized by the presence of autoantibodies in the patient's serum (step 4). The autoantibodies are directed against the cytochrome P450 which generated the metabolite(s). Although the process by which TA and DH induce-hepatitis has been elucidated, further studies are necessary to generalize this mechanism. In addition, an animal model will also be useful to fully understand the immune mechanism of this type of disease.

Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies.

Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor. These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite. The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers. The average level was 69 pmol of CYP1A2/mg of microsomal proteins. Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts.

Specificity of in vitro covalent binding of tienilic acid metabolites to human liver microsomes in relationship to the type of hepatotoxicity: comparison with two directly hepatotoxic drugs.

In order to better understand the first steps leading to drug-induced immunoallergic hepatitis, we studied the target of anti-LKM2 autoantibodies appearing in tienilic acid-induced hepatitis, and the target of tienilic acid-reactive metabolites. It was identified as cytochrome P450 2C9, (P450 2C9): indeed, anti-LKM2 specifically recognized P450 2C9, but none of the other P450s tested (including other 2C subfamily members, 2C8 and 2C18). Tienilic acid-reactive metabolite(s) specifically bound to P450 2C9, and experiments with yeast expressing active isolated P450s showed that P450 2C9 was responsible for tienilic acid-reactive metabolite(s) production. Results of qualitative and quantitative covalent binding of tienilic acid metabolite(s) to human liver microsomes were then compared to those obtained with two drugs leading to direct toxic hepatitis, namely, acetaminophen and chloroform. Kinetic constants (Km and Vmax) were measured, and the covalent binding profile of the metabolites to human liver microsomal proteins was studied. Tienilic acid had both the lowest Km and the highest covalent binding rate at pharmacological doses. For acetaminophen and chloroform, several microsomal proteins were covalently bound, while covalent binding was highly specific for tienilic acid and dihydralazine, another drug leading to immunoallergic hepatitis. Although low numbers of drugs were tested, these results led us to think that there may exist a relationship between the specificity of covalent binding and the type of hepatotoxicity.

Hemoglobin abnormalities. An evaluation on new-born infants and their mothers in a maternity unit close to Brazzaville (P.R. Congo).

In order to evaluate the polymorphism of hemoglobin in a population of Equatorial Africa, we undertook a prospective study of 146 births at a rural maternity hospital close to Brazzaville (P.R. Congo). This showed among the mothers 31 (22%) carriers of the sickle cell trait (AS), six with delta mutation, and two with beta-thalassemia trait. Among the children, 27 (18.5%) had sickle cell trait and one had sickle cell homozygosity. The frequency of the HbF Sardinia trait was 7.5%. This and other studies suggested a dilution gradient from Europe to Africa. Hemoglobin Bart's could be visually detected in 23.3% of the new-born babies. We attempted to distinguish between those infants with a high level of Hb Bart's (Bart's ++ group: 13.7%) and a group with a detectable Hb Bart's level that in our experimental conditions is between 1 and 2% (Bart's + group: 9.6%). Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were 88.6 +/- 5.7 fl and 29.0 +/- 2.1 pg in the Bart's ++ group; 94.5 +/- 10.9 fl and 30.6 +/- 4.1 pg in the Bart's + group; whereas they were 101.0 +/- 8.7 fl and 33.9 +/- 2.5 pg in the control group. Since iron deficiencies are very rare in new-borns and selecting according to published data on black people as homozygous alpha-thalassemia of the type I (-alpha/-alpha), individuals of the Bart's ++ group whose MCV was below 95 fl and MCV below 30 pg, the gene frequency is estimated to be 34% and that of heterozygotes (-alpha/alpha alpha) 45%. These high frequencies were confirmed in AS mothers: 45% showed a significant decrease of the S fraction.