Application of multiblock modelling to identify key drivers for antimicrobial use in pig production in four European countries.

Antimicrobial use in pig farming is influenced by a range of risk factors, including herd characteristics, biosecurity level, farm performance, occurrence of clinical signs and vaccination scheme, as well as farmers' attitudes and habits towards antimicrobial use. So far, the effect of these risk factors has been explored separately. Using an innovative method called multiblock partial least-squares regression, this study aimed to investigate, in a sample of 207 farrow-to-finish farms from Belgium, France, Germany and Sweden, the relative importance of the six above mentioned categories or 'blocks' of risk factors for antimicrobial use in pig production. Four country separate models were developed; they showed that all six blocks provided useful contribution to explaining antimicrobial use in at least one country. The occurrence of clinical signs, especially of respiratory and nervous diseases in fatteners, was one of the largest contributing blocks in all four countries, whereas the effect of the other blocks differed between countries. In terms of risk management, it suggests that a holistic and country-specific mitigation strategy is likely to be more effective. However, further research is needed to validate our findings in larger and more representative samples, as well as in other countries.

Influence of a brisk walking program on postural responses in sedentary older women: a randomised trial.

This study analyzes the evolution in kinematic and non-linear stabilometric parameters in elderly sedentary women selected to participate in a brisk walking program. Ninety-four women were randomly selected for a program of 78 sessions over 6 months, with three sessions of 60 min per week. On the force platform, participants were assessed with both eyes opened as well as eyes closed during a period of 51.2 s and the sampling frequency was 40 Hz. The main dependent kinematic variables were the length, stabilogram surface, and the mean position in anteroposterior as well as medio-lateral directions. For the dynamic approach, we have selected the parameters of recurrence quantification analysis, sample entropy, and multiscale entropy. The kinematic and the time series analysis of group × time interactions demonstrated that 6 months of walk-training lacked influence on kinematic postural responses and on dynamical measurements. The weekly brisk walking program was situated on flat ground and consisted of three 60-min weekly sessions lasting 6 months, leading to no significant effect on postural responses. In regards to international recommendations brisk walking is a pertinent exercise. However, in older sedentary women, our study indicated a systemic lack of influence of 6 months' walk-training on flat ground on kinematic postural responses and on dynamical measures obtained by time series analysis.

Effects of improved early-life conditions on health, welfare, and performance of pigs raised on a conventional farm.

Nowadays, most pigs are raised indoors, on intensive farms providing a poor environment. In these conditions, the risk of the occurrence of damaging behaviours is high, with dramatic consequences for animal health and welfare as well as economic losses for farmers. Early-life conditions may predispose individuals to develop damaging behaviours later in life. In contrast, reinforcing affiliative behaviours between piglets before weaning might help to prevent tail-biting episodes. In this field study, we aimed at improving early-life conditions of piglets on a commercial farm by completely suppressing painful procedures and staggering their exposure to weaning stress factors. The alternative early-life management strategy combined housing in free-farrowing pens with temporary crating of the sow, socialisation during the lactation period with whole-life maintenance of the hierarchical groups, and delayed transfer to the postweaning room after sow removal. Control conditions included birth in farrowing crates, tail docking, absence of socialisation during the lactation period, abrupt weaning with immediate transfer to the postweaning room and mixing with non-littermates. We evaluated the health, welfare, and performance of alternatively raised pigs (n = 80) as compared to controls (n = 75). Visits were made throughout the lifespan of individuals to evaluate their growth and health status. Body and tail lesions were scored as proxy measures of aggressiveness and impaired welfare. Blood and bristle samples were periodically collected to evaluate stress, inflammation and immune competence. While the whole-life performance of pigs was similar among groups, the alternative early-life conditions prevented the growth slowdown usually observed after weaning. In addition, alternatively raised pigs displayed more neutrophils, eosinophils and monocytes the day after weaning, as well as higher C-Reactive Protein levels. One week later, their monocytes displayed greater phagocytic capacity. Altogether, these data suggest an enhanced innate immune competence for alternatively raised pigs around weaning. Piglets reared under alternative conditions also exhibited fewer and less severe body lesions than standard pigs, one week after weaning. In contrast, they showed more tail lesions on days 36 and 66 associated with greater levels of acute phase proteins (C-Reactive Protein and haptoglobin). To conclude, alternative early-life management better prepared piglets for weaning. However, the whole-life maintenance of early-established social groups was not sufficient to prevent the occurrence of damaging behaviours in undocked pigs.

Parent training for the treatment of irritability in children and adolescents: a multisite randomized controlled, 3-parallel-group, evaluator-blinded, superiority trial.

BACKGROUND: Irritability is common in children and adolescents with Attention Deficit Hyperactivity Disorder (ADHD), Oppositional Defiant Disorder (ODD) and with anxiety/depressive disorders. Although youth irritability is linked with psychiatric morbidity, little is known regarding its non-pharmacological treatments. Developing non-pharmacological treatments for children with severe, chronic irritability is an important target for clinical research. To achieve this goal, we will test the benefits of parent-focused therapies in reducing irritability. The aim of the study is to compare Parent Management Training (PMT) and Non-Violent Resistance Training (NVR) programs with treatment-as-usual (TAU) on the improvement of irritability in children and adolescents with a baseline Parent-rated Affective Reactivity Index of 4 or higher, in the context of ADHD and other emotional and behavioural disorders. Additionally, we will assess (i) improvement of irritability at different times and according to different informants (parents, children, clinicians); (ii) improvement of parental strategies; and (iii) acceptability of the interventions, exploring possible mechanisms of the therapeutic effect. METHODS: Two hundred and seventy participants between 6 and 15 years with ADHD and other emotional and behavioural disorders will be recruited and randomly assigned with their parents to the PMT, NVR, and TAU groups. PMT and NVR programs have 10 online sessions and two booster sessions at 1 and at 3 months. The primary outcome measure is the change from baseline at 3 months after completion of the program of the Clinician-rated Affective Rating Scale (CL-ARI) assessed by a blind evaluator. Secondary outcome measures include the change from baseline from those scales: the CL-ARI, the Clinical Global Impression Improvement scale, the Parenting and Familial Adjustment Scales, the Child-rated Cranky thermometers and the Parent-rated ARI. We will assess the parent's expressed emotions and reflexivity during the online five-minute speech sample, clinical dimensions through the Child Behavior Checklist 6-18 and the Inventory of Callous Unemotional traits. Evaluations will be done remotely at baseline and at 1- and 3-months follow-up visits. DISCUSSION: We expect a benefit in controlling irritability in the treatment groups. This will constitute an important achievement in promoting parental support programs in the treatment of irritability in the context of emotional and behavioural disorders. CLINICALTRIALS: gov. Number: NCT05528926. Registered on the 2nd of September, 2022.

Prospecting potential links between PRRSV infection susceptibility of alveolar macrophages and other respiratory infectious agents present in conventionally reared pigs.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is one of the main component of the porcine respiratory disease complex (PRDC), which strongly impact the pig production. Although PRRSV is often considered as a primary infection that eases subsequent respiratory coinfections, the possibility that other PRDC components may facilitate PRRSV infection has been largely overlooked. The main cellular targets of PRRSV are respiratory macrophages among them alveolar macrophages (AM) and pulmonary intravascular macrophages (PIM). AM, contrarily to PIM, are directly exposed to the external respiratory environment, among them co-infectious agents. In order to explore the possibility of a co-infections impact on the capacity of respiratory macrophages to replicate PRRSV, we proceed to in vitro infection of AM and PIM sampled from animals presenting different sanitary status, and tested the presence in the respiratory tract of these animals of the most common porcine respiratory pathogens (PCV2, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma floculare, Pasteurella multocida, Bordetella bronchiseptica, Streptoccocus suis). In this exploratory study with a limited number of animals, no statistic differences were observed between AM and PIM susceptibility to in vitro PRRSV infection, nor between AM coming from animals presenting very contrasting respiratory coinfection loads.

Detection by real-time RT-PCR of a bovine respiratory syncytial virus vaccine in calves vaccinated intranasally.

Seventeen four- to five-week-old calves that were not shedding bovine respiratory syncytial virus (BRSV) were vaccinated intranasally against the disease and sampled by nasal swabbing on 16 different days for up to 20 days after vaccination. BRSV vaccine virus was detected in 15 of the 17 calves. Five of the calves were PCR positive on only one swab, eight were PCR positive on two to five swabs and two were PCR positive on more than five swabs. Twelve of the calves were positive only before day 14 and three were positive after day 14. The nasal shedding of BRSV vaccine virus was very variable.

Guidance on the Selection of Appropriate Indicators for Quantification of Antimicrobial Usage in Humans and Animals.

An increasing variety of indicators of antimicrobial usage has become available in human and veterinary medicine, with no consensus on the most appropriate indicators to be used. The objective of this review is therefore to provide guidance on the selection of indicators, intended for those aiming to quantify antimicrobial usage based on sales, deliveries or reimbursement data. Depending on the study objective, different requirements apply to antimicrobial usage quantification in terms of resolution, comprehensiveness, stability over time, ability to assess exposure and comparability. If the aim is to monitor antimicrobial usage trends, it is crucial to use a robust quantification system that allows stability over time in terms of required data and provided output; to compare usage between different species or countries, comparability must be ensured between the different populations. If data are used for benchmarking, the system comprehensiveness is particularly crucial, while data collected to study the association between usage and resistance should express the exposure level and duration as a measurement of the exerted selection pressure. Antimicrobial usage is generally described as the number of technical units consumed normalized by the population at risk of being treated in a defined period. The technical units vary from number of packages to number of individuals treated daily by adding different levels of complexity such as daily dose or weight at treatment. These technical units are then related to a description of the population at risk, based either on biomass or number of individuals. Conventions and assumptions are needed for all of these calculation steps. However, there is a clear lack of standardization, resulting in poor transparency and comparability. By combining study requirements with available approaches to quantify antimicrobial usage, we provide suggestions on the most appropriate indicators and data sources to be used for a given study objective.

Herd-specific interventions to reduce antimicrobial usage in pig production without jeopardising technical and economic performance.

Pig farmers are strongly encouraged to reduce their antimicrobial usage in order to reduce the risk of antimicrobial resistance. Herd-level intervention is needed to achieve national and European reduction targets. Alternative, especially preventive measures, have to be implemented to reduce the need for antimicrobial treatments. However, little is known about the feasibility, effectiveness and return on investment of such measures. The objective of this study was to assess, across four countries, the technical and economic impact of herd-specific interventions aiming at reducing antimicrobial usage in pig production while implementing alternative measures. An intervention study was conducted between February 2014 and August 2015 in 70 farrow-to-finish pig farms located in Belgium, France, Germany and Sweden. Herd-specific interventions were defined together with the farmer and the herd veterinarian. Farms were followed over one year and their antimicrobial usage and technical performance were compared with values from the year before intervention. Compliance with the intervention plan was also monitored. Changes in margin over feed cost and net farm profit were estimated in a subset of 33 Belgian and French farms with sufficient data, using deterministic and stochastic modeling. Following interventions, a substantial reduction in antimicrobial use was achieved without negative impact the overall farm technical performance. A median reduction of 47.0% of antimicrobial usage was achieved across four countries when expressed in terms of treatment incidence from birth to slaughter, corresponding to a 30.5% median reduction of antimicrobial expenditures. Farm compliance with intervention plans was high (median: 93%; min-max: 20; 100) and farms with higher compliance tended to achieve bigger reduction (ρ=-0.18, p=0.162). No association was found between achieved reduction and type or number of alternative measures implemented. Mortality in suckling piglets, weaners and fatteners, daily weight gain and feed conversion ratio did not significantly change over the course of the study, while the number of weaned piglets per sow per year slightly increased. The median change in net farm profit among Belgian and French farms was estimated to be €4.46 (Q25-Q75:-32.54; 80.50) and €1.23 (Q25-Q75:-32.55; 74.45) per sow per year using the detererministic and stochastic models, respectively. It was more influenced by a change in feed conversion ratio and daily weight gain than by a change in antimicrobial expenditures or intervention direct net cost. Therefore, costs of alternative measures should not be perceived as a barrier, but rather as an opportunity to optimise production practices for sustained productivity and improved animal health.

Quantitative and qualitative antimicrobial usage patterns in farrow-to-finish pig herds in Belgium, France, Germany and Sweden.

Data on sales of antimicrobials using a standardised methodology have shown that there are vast differences between countries in amounts of antimicrobials sold for food-producing animals, but these data do not provide insight on how sales are distributed by species and age groups. The aim of this study was to compare herd level antimicrobial usage for pigs by age category, antimicrobial class and administration route for farrow-to-finish herds in four EU countries. A cross-sectional study was conducted among 227 farrow-to-finish pig herds with at least 100 sows and 500 finishing pigs in Belgium (n=47), France (n=60), Germany (n=60) and Sweden (n=60). Detailed information about the antimicrobial consumption for breeding and growing pigs was collected. Antimicrobial usage was quantified as active substance expressed as mg and then converted to treatment incidence (TI) based on Defined Daily Doses Animal per 1000 pig-days at risk. TIs varied between and within countries, herds and age groups. The Swedish herds had the lowest and the German herds the highest overall use. Most treatments were applied to weaned piglets except in the Swedish herds where treatments of suckling piglets were most frequent. Antimicrobials were most often applied through feed or water except in the Swedish herds where parenteral treatments were most frequent. Aminopenicillins was the antimicrobial class most commonly used. Use of third and fourth generation cephalosporins constituted 11% of use for the Belgian herds, which was higher compared to the other countries. There was a significant (p<0.01) association between the within-herd antimicrobial use across different age categories. This study has shown that there were large differences in antimicrobial use for pigs between countries, herds and age groups in farrow-to-finish herds of similar size when actual consumption data were compared. Collecting detailed usage data can be used to efficiently target high users in order to reduce antimicrobial consumption.

The biosecurity status and its associations with production and management characteristics in farrow-to-finish pig herds.

Disease prevention through biosecurity measures is believed to be an important factor for improvement of the overall health status in animal production. This study aimed at assessing the levels of implementation of biosecurity measures in pig production in four European Union (EU) countries and to describe possible associations between the biosecurity level and farm and production characteristics. A cross-sectional study was conducted in 232 farrow-to-finish pig herds in Belgium, France, Germany and Sweden between December 2012 and December 2013. The biosecurity status in each of these herds was described and quantified by using the risk-based scoring tool Biocheck.UGentTM (www.biocheck.ugent.be). Production and management characteristics, obtained from the herd management system and by interviewing the farmer, were analysed for their association with the biosecurity level. A causal path was designed to study statistical associations. The results showed that there was substantial room for improvement in the biosecurity status on many pig farms. Significant differences (P<0.01) both in internal and external biosecurity levels were observed between countries. The external biosecurity status, combining all measures taken to prevent disease introduction into the herd, was highest in Germany and lowest in France. The internal biosecurity status, combining all measures taken to prevent within herd disease transmission, was highest in Sweden and lowest, with a large variation, in Belgium. External biosecurity scores were in general higher compared to internal biosecurity scores. The number of pathogens vaccinated against was significantly associated with internal biosecurity status, suggesting an overall more preventive approach towards the risk of disease transmission. A higher external biosecurity was associated with more weaned piglets per sow per year. Furthermore also the weaning age and the mortality till weaning were highly associated with the number of weaned piglets per sow per year. The negative association observed between the biosecurity level and the estimated frequency of treatment against certain clinical signs of disease as a proxy for disease incidence is consistent with the hypothesis that a higher biosecurity level results in healthier animals. These findings promote an improved biosecurity status at pig farms and are of relevance in the discussion on alternative ways to keep animals healthy with a reduced necessity of antimicrobials; Prevention is better than cure!

A Comparison of Pig Farmers' and Veterinarians' Perceptions and Intentions to Reduce Antimicrobial Usage in Six European Countries.

Antimicrobial (AM) resistance is an increasing problem in human and veterinary medicine. To manage this problem, the usage of AM should be reduced in pig farming, as well as in other areas. It is important to investigate the factors that influence both pig farmers' and veterinarians' intentions to reduce AM usage, which is a prerequisite for developing intervention measures. We conducted a mail survey among pig farmers (N = 1,294) and an online survey among veterinarians (N = 334) in Belgium, Denmark, France, Germany, Sweden and Switzerland. The farmers' survey assessed the perceived risks and benefits of and need for AM usage; the intention to reduce AM usage; farmers' efficacy (i.e. perception of their ability to reduce AM usage); support from their veterinarian; and the future reduction potential of AM usage. Additionally, self-reported reduction behaviours, the perceived farmers' barriers to reduce AM usage and relationships with farmers were assessed in the veterinarians' survey. The results showed that farmers and veterinarians had similar perceptions of the risks and benefits of AM usage. Veterinarians appeared to be more optimistic than pig farmers about reducing AM usage in pig farming. Farmers believed that their efficacy over AM reduction was relatively high. Farmers' intention to reduce AM usage and veterinarians' self-reported reduction behaviours were mainly associated with factors concerning the feasibility of reducing AM usage. To promote prudent AM usage, pig farmers should learn and experience how to reduce usage by applying alternative measures, whereas veterinarians should strengthen their advisory role and competencies to support and educate farmers.

Higher perceived risk of antimicrobials is related to lower antimicrobial usage among pig farmers in four European countries.

The prudent use of antimicrobials (AMs) should be widened in pig farming to reduce the risk of AM resistance (AMR) in human and veterinary medicine. It is therefore important to understand pig farmers' motivators and the barriers to AM usage (AMU) on their farms. The authors investigated pig farmers' self-estimated levels of AMU, their perceived benefits and risks and the need for AMs in a cross-sectional survey in Belgium, France, Germany and Sweden. The authors also compared these perceptions between the four countries and related them to pig farmers' actual AMU. The results showed that farmers who used more AMs also estimated their own usage as higher. Farmers perceived many benefits but relatively few risks of AMU in pig farming. Some significant cross-country differences in farmers' perceptions were found, but they were relatively small. After controlling for country differences and farm differences, only perceived risks had a significant association with AMU. The authors therefore conclude that in order to promote prudent AMU, it seems most promising to focus on the structural differences in pig farming and veterinary medicine (e.g. legislation, role of the veterinarian) among countries. In addition, interventions which aim at reducing AMU should increase farmers' awareness of the risks of extensive AMU.

Review: Towards the agroecological management of ruminants, pigs and poultry through the development of sustainable breeding programmes. II. Breeding strategies.

Agroecology uses ecological processes and local resources rather than chemical inputs to develop productive and resilient livestock and crop production systems. In this context, breeding innovations are necessary to obtain animals that are both productive and adapted to a broad range of local contexts and diversity of systems. Breeding strategies to promote agroecological systems are similar for different animal species. However, current practices differ regarding the breeding of ruminants, pigs and poultry. Ruminant breeding is still an open system where farmers continue to choose their own breeds and strategies. Conversely, pig and poultry breeding is more or less the exclusive domain of international breeding companies which supply farmers with hybrid animals. Innovations in breeding strategies must therefore be adapted to the different species. In developed countries, reorienting current breeding programmes seems to be more effective than developing programmes dedicated to agroecological systems that will struggle to be really effective because of the small size of the populations currently concerned by such systems. Particular attention needs to be paid to determining the respective usefulness of cross-breeding v. straight breeding strategies of well-adapted local breeds. While cross-breeding may offer some immediate benefits in terms of improving certain traits that enable the animals to adapt well to local environmental conditions, it may be difficult to sustain these benefits in the longer term and could also induce an important loss of genetic diversity if the initial pure-bred populations are no longer produced. As well as supporting the value of within-breed diversity, we must preserve between-breed diversity in order to maintain numerous options for adaptation to a variety of production environments and contexts. This may involve specific public policies to maintain and characterize local breeds (in terms of both phenotypes and genotypes), which could be used more effectively if they benefited from the scientific and technical resources currently available for more common breeds. Last but not least, public policies need to enable improved information concerning the genetic resources and breeding tools available for the agroecological management of livestock production systems, and facilitate its assimilation by farmers and farm technicians.

Review: Towards the agroecological management of ruminants, pigs and poultry through the development of sustainable breeding programmes: I-selection goals and criteria.

Agroecology uses natural processes and local resources rather than chemical inputs to ensure production while limiting the environmental footprint of livestock and crop production systems. Selecting to achieve a maximization of target production criteria has long proved detrimental to fitness traits. However, since the 1990s, developments in animal breeding have also focussed on animal robustness by balancing production and functional traits within overall breeding goals. We discuss here how an agroecological perspective should further shift breeding goals towards functional traits rather than production traits. Breeding for robustness aims to promote individual adaptive capacities by considering diverse selection criteria which include reproduction, animal health and welfare, and adaptation to rough feed resources, a warm climate or fluctuating environmental conditions. It requires the consideration of genotype×environment interactions in the prediction of breeding values. Animal performance must be evaluated in low-input systems in order to select those animals that are adapted to limiting conditions, including feed and water availability, climate variations and diseases. Finally, we argue that there is no single agroecological animal type, but animals with a variety of profiles that can meet the expectations of agroecology. The standardization of both animals and breeding conditions indeed appears contradictory to the agroecological paradigm that calls for an adaptation of animals to local opportunities and constraints in weakly artificialized systems tied to their physical environment.

Perceptions of antimicrobial usage, antimicrobial resistance and policy measures to reduce antimicrobial usage in convenient samples of Belgian, French, German, Swedish and Swiss pig farmers.

We conducted a survey among convenient samples of pig farmers (N=281) in Belgium, France, Germany, Sweden and Switzerland. We identified some significant differences among the five investigated countries (independent variable) regarding farmers' antimicrobial usage compared to their own country and worries related to pig farming (dependent variables), but most of the differences were rather small. In general, farmers perceived their own antimicrobial usage to be lower than that of their peers in the same country and lower than or similar to that of farmers from other countries. This may be a consequence of our convenience sample, resulting in self-selection of highly motivated farmers. Farmers were significantly more worried about financial/legal issues than about antimicrobial resistance. They believed that a reduction in revenues for slaughter pigs treated with a large amount of antimicrobials would have the most impact on reduced antimicrobial usage in their country. Further, farmers who were more worried about antimicrobial resistance and who estimated their own antimicrobial usage as lower than their fellow countrymen, perceived more impact from policy measures on the reduction of antimicrobials. Our results indicated that the same policy measures can be applied to reduce antimicrobial usage in pig farming in all five countries. Moreover, it seems worthwhile to increase pig farmers' awareness of the threat of antimicrobial resistance and its relation to antimicrobial usage; not only because pig farmers appeared little worried about antimicrobial usage but also because it affected farmers' perception of policy measures to reduce antimicrobial usage. Our samples were not representative for the national pig farmer populations. Further research is therefore needed to examine to what extent our findings can be generalised to these populations and to farmers in other countries.

Epitope mapping of human CYP1A2 in dihydralazine-induced autoimmune hepatitis.

Dihydralazine-induced hepatitis is characterized by the presence of anti-liver microsomal (anti-LM) autoantibodies in the sera of patients. Cytochrome P450 1A2 (CYP1A2), involved in the metabolism of dihydralazine, was shown to be a target for autoantibodies. In order to investigate further the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, and since the specificity of anti-LM autoantibodies towards CYP1A2 has been determined, the antigenic site was further localized. By constructing fragments derived from CYP1A2 cDNA and probing the corresponding proteins with several anti-LM sera, we were able to define a region (amino acid 335-471) which was immunoreactive with 100% of sera. An internal deletion in this region led to the loss of recognition by anti-LM autoantibodies, confirming that the epitope was conformational. Epitope mapping studies had previously been performed for CYP2D6, CYP17, CYP21A2, and recently for CYP3A1 and CYP2C9. Those data were compared with results obtained in the present study for CYP1A2.

Human cytochrome P450 2E1 (CYP2E1): from genotype to phenotype.

CYP2E1 is involved in the activation of various carcinogens, including N-nitrosamines, which are believed to be important in human carcinogenesis. Humans exhibit wide interindividual variability in levels of CYP2E1 mRNA and protein, which might explain interindividual differences in susceptibility to carcinogens activated by CYP2E1. Such variability could be due either to genetic polymorphisms observed in the CYP2E1 gene (Rsa I in the 5'-flanking region, Dra I in intron 6 and Taq I in intron 7) or to varying inducibility by xenobiotics. The aim of the present study was to establish whether, in a Caucasian population (n = 93), there existed a relationship between allelic forms of the CYP2E1 gene and the phenotype determined in vitro by hepatic ability to 6-hydroxylate chlorzoxazone. Rates of chlorzoxazone-6-hydroxylation were significantly correlated with levels of immunochemically measured CYP2E1 (p < 0.001). CYP1A2, 2C8, 2C9, 2C18, 2D6, 3A4 and 3A5 did not appear to be significantly involved in chlorzoxazone metabolism, whereas the participation of CYP1A1 could not be excluded. Frequencies of the rare alleles for the three polymorphism sites were 2.2% for RsaI, 7.5% for DraI and 8.5% for TaqI. Despite substantial interindividual variations in chlorzoxazone hydroxylase activity, no relationship between any of the three polymorphisms and CYP2E1 activity was established. Therefore, in humans, interindividual variability in CYP2E1 levels is probably due to differing induction levels as a result of environmental factors, or to genetic factors other than those studied in this work.

Evaluation of caffeine as a test drug for CYP1A2, NAT2 and CYP2E1 phenotyping in man by in vivo versus in vitro correlations.

Caffeine is used to phenotype subjects in vivo for the cytochrome P450 isoforms CYP1A2 and CYP2E1, and for N-acetyltransferase type 2 (NAT2). However, how much of the variation in phenotyping parameters may be attributed to variations in CYP1A2 and CYP2E1 activities has not been determined. Therefore, this study intraindividually compared enzyme activities and/or content in liver samples with pharmacokinetic parameters of caffeine in vivo after administration of a test dose in 25 patients undergoing hepatectomy. Parameters measured in vitro were the high affinity components of caffeine 3-demethylation and phenacetin 0-deethylation, microsomal CYP1A2 and CYP2E1 immunoreactivity, and cytosolic sulfamethazine N-acetylation. Caffeine parameters in vivo included caffeine clearance from plasma and/or saliva, paraxanthine/caffeine ratios in plasma and saliva, plasma theophylline/caffeine ratio, and several metabolite ratios from spot urine sampled 6 h postdose. Correlations between parameters were determined using weighted linear regression analyses. Caffeine clearance and paraxanthine/caffeine ratios correlated most highly to intrinsic clearance of caffeine 3-demethylation and to CYP1A2 immunoreactivity (r= 0.584-0.82), whereas urinary CYP1A2 ratios correlated less strongly with CYP1A2 parameters in vitro. Assignment of acetylator phenotype by urinary NAT2 ratios was concordant with sulfamethazine-N-acetylation in vitro. In contrast to CYP1A2 parameters in vitro, CYP2E1 immunoreactivity was not related to the theophylline/caffeine plasma ratio. CYP1A2 activity, thus, is the major determinant of caffeine clearance and the paraxanthine/caffeine ratios in vivo, of which the saliva ratio 6 h postdose appears as the most advantageous parameter. The results confirm that phenotyping using caffeine provides valid estimates of CYP1A2 and NAT2 activity.

Involvement of cytochrome P450 3A enzyme family in the major metabolic pathways of toremifene in human liver microsomes.

The anti-estrogen toremifen-Fc-1157a or 4-chloro-1,2-diphenyl-1-[4-[2(N,N-dimethylamino)ethoxy]-phenyl]-1- butene is now used for the treatment of breast cancer. This drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-demethyl-(DMTOR), 4-hydroxy-(4OH-TOR) and deamino-hydroxy-(TOR III) toremifene metabolites. The specific forms of cytochrome P450 involved in these oxidation reactions were examined in 32 human liver microsomal preparations previously characterized with respect to their contents of several known P450 enzymes. Toremifene was demethylated with an apparent Km of 124 microM while it was hydroxylated with an apparent Km of 139 microM. The metabolic rates were 71 +/- 56, 13 +/- 9 and 15 +/- 4 pmol/min/mg microsomal protein, respectively, for DMTOR, 4-OH-TOR and TOR III. The N-demethylation activity was strongly correlated with estradiol 2-hydroxylation (r = 0.75), nifedipine oxidation (r = 0.86), tamoxifen N-demethylation (r = 0.73), testosterone 6 beta-hydroxylation (r = 0.78) and erythromycin N-demethylation (r = 0.84), all these monooxygenase activities known to be supported by CYP3A4 isoform. Furthermore, the CYP3A content of liver microsomal samples, measured by western blot analysis using a monoclonal anti-human CYP3A4 antibody, was strongly correlated with DMTOR formation (r = 0.80). Compounds such as cyclosporin, triacetyl-oleandomycin and testosterone inhibited the N-demethylation of toremifene metabolism at 80, 89 and 56% vs control, respectively, while the formation of TOR III was inhibited at 78, 82 and 73% vs control and the 4-hydroxylation pathway was inhibited no more than about 50% vs control. Prior incubation of microsomes with 100 microM gestodene, known to be a selective mechanism-based inhibitor of CYP3A4 in the presence of NADPH, led to 76 +/- 6 and 76 +/- 5% (N = 5 samples) reductions in the N-demethylation and formation of TOR III, respectively. Polyclonal antibody directed against human CYP3A enzymes inhibited formation of DMTOR and TOR III by 60 and 46%, respectively. The metabolism of toremifene was not activated by alpha-naphthoflavone. Finally, the use of yeasts genetically engineered for expression of human P4501A1, 1A2, 2C9 and 3A4 allowed us to demonstrate that DMTOR and TOR III formations are mediated by P4501A and 3A4 enzymes and by contrast these enzymes are not involved in the 4-hydroxylation pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical localization of cytochrome P450 2E1 in human pulmonary carcinoma and normal bronchial tissue.

Cytochrome P450 2E1 (CYP2E1) is a major xenobiotic-metabolizing enzyme but data concerning its extrahepatic expression are few. CYP2E1 can metabolically activate many procarcinogens and therefore its presence in the lung might play a role in bioactivation of procarcinogens, so we studied the expression and localization of CYP2E1 in primary pulmonary carcinomas and surrounding normal bronchial tissue from 28 patients. Seromucous glands showed expression of CYP2E1 in 19 and bronchial epithelium in 18 of the 28 samples of normal bronchial tissue. Thirteen of the corresponding cases of primary pulmonary carcinoma showed staining for CYP2E1. In 11 of these 13 cases, CYP2E1 was also present in normal bronchial tissue. There was no statistically significant difference in the expression of CYP2E1 between adenocarcinomas and squamous cell carcinomas. No association was observed between the expression of CYP2E1 in tumour tissue and normal bronchial tissue. However, there was a significant correlation between the expression of CYP2E1 in seromucous glands and bronchial epithelium (r = 0.61, P < 0.01) of normal tissue. We conclude that CYP2E1 can be present in both normal and neoplastic bronchial tissue.