Human Variability in Carboxylesterases and carboxylesterase-related Uncertainty Factors for Chemical Risk Assessment.

Carboxylesterases (CES) are an important class of enzymes involved in the hydrolysis of a range of chemicals and show large inter-individual variability in vitro. An extensive literature search was performed to identify in vivo probe substrates for CES1 and CES2 together with their protein content and enzymatic activity. Human pharmacokinetic (PK) data on Cmax, clearance, and AUC were extracted from 89 publications and Bayesian meta-analysis was performed using a hierarchical model to derive CES-related variability distributions and related uncertainty factors (UF). The CES-related variability indicated that 97.5% of healthy adults are covered by the kinetic default UF (3.16), except for clopidogrel and dabigatran etexilate. Clopidogrel is metabolised for a small amount by the polymorphic CYP2C19, which can have an impact on the overall pharmacokinetics, while the variability seen for dabigatran etexilate might be due to differences in the absorption, since this can be influenced by food intake. The overall CES-related variability was moderate to high in vivo (

Human variability in polymorphic CYP2D6 metabolism: Implications for the risk assessment of chemicals in food and emerging designer drugs.

The major human cytochrome P450 CYP2D6 isoform enzyme plays important roles in the liver and in the brain with regards to xenobiotic metabolism. Xenobiotics as CYP2D6 substrates include a whole range of pharmaceuticals, pesticides and plant alkaloids to cite but a few. In addition, a number of endogenous compounds have been shown to be substrates of CYP2D6 including trace amines in the brain such as tyramine and 5-methoxytryptamine as well as anandamide and progesterone. Because of the polymorphic nature of CYP2D6, considerable inter-phenotypic and inter-ethnic differences in the pharmaco/toxicokinetics (PK/TK) and metabolism of CYP2D6 substrates exist with potential consequences on the pharmacology and toxicity of chemicals. Here, large extensive literature searches have been performed to collect PK data from published human studies for a wide range of pharmaceutical probe substrates and investigate human variability in CYP2D6 metabolism. The computed kinetic parameters resulted in the largest open source database, quantifying inter-phenotypic differences for the kinetics of CYP2D6 probe substrates in Caucasian and Asian populations, to date. The database is available in supplementary material (CYPD6 DB) and EFSA knowledge junction (DOI to added). Subsequently, meta-analyses using a hierarchical Bayesian model for markers of chronic oral exposure (oral clearance, area under the plasma concentration time curve) and acute oral exposure (maximum plasma concentration (Cmax) provided estimates of inter-phenotypic differences and CYP2D6-related uncertainty factors (UFs) for chemical risk assessment in Caucasian and Asian populations classified as ultra-rapid (UM), extensive (EMs), intermediate (IMs) and poor metabolisers (PMs). The model allowed the integration of inter-individual (i.e. inter-phenotypic and inter-ethnic), inter-compound and inter-study variability together with uncertainty in each PK parameter. Key findings include 1. Higher frequencies of PMs in Caucasian populations compared to Asian populations (>8% vs 1-2%) for which EM and IM were the most frequent phenotype. 2. Large inter-phenotypic differences in PK parameters for Caucasian EMs (coefficients of variation (CV) > 50%) compared with Caucasian PMs and Asian EMs and IMs (i.e CV < 40%). 3. Inter-phenotypic PK differences between EMs and PMs in Caucasian populations increase with the quantitative contribution of CYP2D6 for the metabolism (fm) for a range of substrates (fmCYP2D6 range: 20-95% of dose) (range: 1-54) to a much larger extent than those for Asian populations (range: 1-4). 4. Exponential meta-regressions between FmCYP2D6 in EMs and inter-phenotypic differences were also shown to differ between Caucasian and Asian populations as well as CYP2D6-related UFs. Finally, implications of these results for the risk assessment of food chemicals and emerging designer drugs of public health concern, as CYP2D6 substrates, are highlighted and include the integration of in vitro metabolism data and CYP2D6-variability distributions for the development of quantitative in vitro in vivo extrapolation models.

Human variability in isoform-specific UDP-glucuronosyltransferases: markers of acute and chronic exposure, polymorphisms and uncertainty factors.

UDP-glucuronosyltransferases (UGTs) are involved in phase II conjugation reactions of xenobiotics and differences in their isoform activities result in interindividual kinetic differences of UGT probe substrates. Here, extensive literature searches were performed to identify probe substrates (14) for various UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15) and frequencies of human polymorphisms. Chemical-specific pharmacokinetic data were collected in a database to quantify interindividual differences in markers of acute (Cmax) and chronic (area under the curve, clearance) exposure. Using this database, UGT-related uncertainty factors were derived and compared to the default factor (i.e. 3.16) allowing for interindividual differences in kinetics. Overall, results show that pharmacokinetic data are predominantly available for Caucasian populations and scarce for other populations of different geographical ancestry. Furthermore, the relationships between UGT polymorphisms and pharmacokinetic parameters are rarely addressed in the included studies. The data show that UGT-related uncertainty factors were mostly below the default toxicokinetic uncertainty factor of 3.16, with the exception of five probe substrates (1-OH-midazolam, ezetimibe, raltegravir, SN38 and trifluoperazine), with three of these substrates being metabolised by the polymorphic isoform 1A1. Data gaps and future work to integrate UGT-related variability distributions with in vitro data to develop quantitative in vitro-in vivo extrapolations in chemical risk assessment are discussed.

Human variability in influx and efflux transporters in relation to uncertainty factors for chemical risk assessment.

Transporters are divided into the ABC and SLC super-families, mediating the cellular efflux and influx of various xenobiotic and endogenous substrates. Here, an extensive literature search was performed to identify in vivo probe substrates for P-gp, BCRP and OAT1/3. For other transporters (e.g. OCT, OATP), no in vivo probe substrates could be identified from the available literature. Human kinetic data (Cmax, clearance, AUC) were extracted from 142 publications and Bayesian meta-analyses were performed using a hierarchical model to derive variability distributions and related uncertainty factors (UFs). For P-gp, human variability indicated that the kinetic default UF (3.16) would cover over 97.5% of healthy individuals, when considering the median value, while the upper confidence interval is exceeded. For BCRP and OAT1/3 human variability indicated that the default kinetic UF would not be exceeded while considering the upper confidence interval. Although limited kinetic data on transporter polymorphisms were available, inter-phenotypic variability for probe substrates was reported, which may indicate that the current default kinetic UF may be insufficient to cover such polymorphisms. Overall, it is recommended to investigate human genetic polymorphisms across geographical ancestry since they provide more robust surrogate measures of genetic differences compared to geographical ancestry alone. This analysis is based on pharmaceutical probe substrates which are often eliminated relatively fast from the human body. The transport of environmental contaminants and food-relevant chemicals should be investigated to broaden the chemical space of this analysis and assess the likelihood of potential interactions with transporters at environmental concentrations.

Bayesian meta-analysis of inter-phenotypic differences in human serum paraoxonase-1 activity for chemical risk assessment.

Human variability in paraoxonase-1 (PON1) activities is driven by genetic polymorphisms that affect the internal dose of active oxons of organophosphorus (OP) insecticides. Here, an extensive literature search has been performed to collect human genotypic frequencies (i.e. L55M, Q192R, and C-108T) in subgroups from a range of geographical ancestry and PON1 activities in three probe substrates (paraoxon, diazoxon and phenyl acetate). Bayesian meta-analyses were performed to estimate variability distributions for PON1 activities and PON1-related uncertainty factors (UFs), while integrating quantifiable sources of inter-study, inter-phenotypic and inter-individual differences. Inter-phenotypic differences were quantified using the population with high PON1 activity as the reference group. Results from the meta-analyses provided PON1 variability distributions and these can be implemented in generic physiologically based kinetic models to develop quantitative in vitro in vivo extrapolation models. PON1-related UFs in the Caucasian population were above the default toxicokinetic UF of 3.16 for two specific genotypes namely -108CC using diazoxon as probe substrate and, -108CT, -108TT, 55MM and 192QQ using paraoxon as probe substrate. However, integration of PON1 genotypic frequencies and activity distributions showed that all UFs were within the default toxicokinetic UF. Quantitative inter-individual differences in PON1 activity are important for chemical risk assessment particularly with regards to the potential sensitivity to organophosphates' toxicity.

Testosterone autoregulation of its biosynthesis in the rat testis: inhibition of 17 alpha-hydroxylase activity.

Using the in vitro perfused rat testis, the effects of testosterone (T) on its own biosynthesis, and in particular on the inhibition of specific steroidogenic step(s) in the biosynthetic pathway from cholesterol to T, were examined. Rat testes perfused in vitro for 1 hour with medium containing 1.5 microM T secreted significantly less T than control testes in response to physiologic or maximal luteinizing hormone (LH) stimulation. To locate the site(s) of this rapid inhibition, the effects of arterial T infusion on steroidogenesis by testes also infused with pregnenolone (PREG), progesterone (PROG), 17 alpha-hydroxyprogesterone (17-PROG), or androstenedione (ADIONE) were measured by summing all the possible reaction products from each substrate. This approach allowed us to examine the effect of T in situ on the reactions: LH-stimulated PREG secretion; PREG to PROG; PROG to 17-PROG; 17-PROG to ADIONE; and ADIONE to T. Only PROG to 17-PROG (17 alpha-hydroxylase activity) was inhibited by arterial T infusion. A kinetic examination of the PROG to 17-PROG reaction demonstrated that the specific inhibition by T was competitive. The apparent km for PROG in this system was 16.0 microM, whereas the apparent ki of T was 1.6 microM, indicating a relatively high degree of sensitivity of the reaction to T. Taken together, these data confirm that T is able to regulate its own synthesis and indicate that this autoregulation is the result of rapid, specific inhibition by T of 17 alpha-hydroxylase activity.

Neuroendocrine responses to social regulation of puberty in the female house mouse.

First estrus is advanced in female house mice exposed to an adult male and delayed in those housed in groups. Experiments were conducted to explore possible mechanisms by which the hypothalamus integrates these puberty-regulating social signals. Female mice weaned at 21 days of age were placed in groups of 8 (G8JF), a juvenile female with a juvenile male (JFJM) or juvenile female with an adult male (JFAM). All females were ovariectomized on day 28 and sacrificed on day 29. There was no significant difference between treatments in the postovariectomy rise in LH. Next, female mice were weaned, assigned to G8JF, JFJM or JFAM treatments and ovariectomized on day 22. Females were sacrificed on day 29, 3 h after injection with either 1.0 micrograms of estradiol, or vehicle. Estradiol significantly suppressed LH in all three treatments, with no differences between treatments. Two-way ANOVA (social treatment x estradiol treatment) revealed no differences or interactions in brain catecholamines as a result of estradiol injection. The G8JF treatment significantly increased norepinephrine, (NE), dopamine (DA) and its metabolite 3,4-dihydroxy-phenylacetic acid in the mediobasal hypothalamus (MBH), and the 3-methoxy-4-hydroxy-phenylglycol/NE ratio in the preoptic area (POA). In the final experiments, isolate prepubertal female mice were treated with either water or male urine (MU) on the oronasal groove. Eight days of MU treatment resulted in significant uterine growth, however there were no differences in serum LH, POA or MBH catecholamines or POA and median eminence LHRH. One hour after application of MU, serum LH was significantly elevated, however, there were no differences in accessory olfactory bulb catecholamines. These results suggest that the mechanism by which male and grouped female exposure alters first estrus may not involve changes in sensitivity to estradiol negative feedback. Grouping may delay first estrus through the negative effects of DA on the LHRH system in the MBH. We observed no differences that might help to explain how male exposure causes LH release.

Behavioral effects in the rat of dihydrexidine, a high-potency, full-efficacy D1 dopamine receptor agonist.

Dihydrexidine (trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phena nthridine) was reported recently to be the first full efficacy, potent D1 receptor agonist, but one also having some potency for D2 receptors. This study reports the effects of dihydrexidine on behavior of the rat. In study 1, the dose-response relationships of dihydrexidine (0.3 to 30 mg/kg) to various behaviors were assessed using direct observations. The frequency of three behaviors (grooming, sniffing, and locomotion) was significantly increased by this drug. The dose-response curve for drug-induced grooming approximated an inverted U shape. Dihydrexidine increased locomotion at two of the higher doses (3 and 30 mg/kg), and increased sniffing at doses greater than or equal to 1.0 mg/kg. Other behavioral topographies, such as licking, gnawing, and rearing, were not systematically affected by drug administration. Also, there was no indication of convulsion in any dihydrexidine-treated rat. In study 2, rats were pretreated with either the selective D1 antagonist SCH23390 or the selective D2 antagonist remoxipride prior to receiving dihydrexidine. SCH23390 antagonized the effects of dihydrexidine on grooming, locomotion, and sniffing. Conversely, remoxipride blocked dihydrexidine-induced locomotion, but had no effect on dihydrexidine-induced grooming or sniffing. Numerous behaviors are believed to be mediated by the interactions of D1 and D2 receptors. These data indicate that dihydrexidine can be an important tool for characterizing both the behavioral actions of D1 receptors, and the nature of D1/D2 interactions in mammalian brain. In addition, its high potency and full efficacy at D1 receptors, coupled with its significant D2 properties, may provide specific utility in certain clinical situations.

Testis function in rhesus monkeys treated with a contraceptive steroid formulation.

Rhesus monkeys (Macaca mulatta) were treated with testosterone (100 micrograms/kg/day) plus estradiol (0.5 micrograms/kg/day) via subcutaneous polydimethylsiloxane (PDS;Silastic) implants for thirteen months. This steroid regimen inhibited LH but not FSH secretion by the anterior pituitary, inhibited testicular testosterone and estradiol production and drastically reduced the numbers of vigorously motile spermatozoa in the ejacula. Importantly, these effects on testis function were achieved at dosages of testosterone and estradiol which approximate the amount of these two steroids produced daily in normal rhesus males.

Simultaneous measurement of four testicular delta 4-3-ketosteroids by isocratic high-performance liquid chromatography with on-line ultraviolet absorbance detection.

A method is described for the simultaneous measurement of testosterone, androstenedione, 17 alpha-hydroxyprogesterone and progesterone in venous effluent from in vitro perfused rat testes. The assay uses a non-radioisotopic internal standard (11 beta-hydroxyandrostenedione), isocratic high-performance liquid chromatography (HPLC) and UV absorbance detection at 240 nm. Either of two isocratic HPLC systems described in this report (tetrahydrofuran-methanol-water, 16:28:56; methanol-acetonitrile-water, 9:36:55; v/v/v) may be used, and assay specificity is the same in each. The separation and measurement of all four steroids are completed in 25 min. Sensitivity of the method is 10 ng for testosterone, androstenedione and 17 alpha-hydroxyprogesterone and 25 ng for progesterone. The linear range of the assay extends through 1600 ng which is the upper amount of each steroid tested. Average inter-assay coefficient of variation was 3.3% and average intra-assay coefficient of variation was 3.6%. This rapid, specific and reliable method requires minimal sample preparation and may be performed by inexperienced personnel.

Analysis of the estrous cycle of the laboratory-housed Senegal galago (Galago senegalensis senegalensis): natural and induced cycles.

The natural estrous cycle of captive Senegal galagos was analyzed from daily records of 11 cycling females for 18 months, and induction of vaginal estrus and ovulation by gonadotropins and estrogen were examined in 9 females. No seasonal trend in cycling was indicated, as at least 5 of the 11 cycling females exhibited vaginal estrus during each month. Cycle lengths and duration of estrus were consistent for each female but varied significantly among females. Individuals' average cycle lengths ranged from 29.2 +/- 0.8 to 39.3 +/- 3.4 days, and duration of estrus from 4.8 +/- 0.2 to 6.7 +/- 0.4 days. Vaginal estrus was induced by PMSG and PMSG-HCG combinations, and by estradiol. The gonadotropins also induced growth and ovulation of more than one follicle.

Autoregulation of testosterone secretion in perfused rat testes.

Infusion of testosterone into the testicular artery, at concentrations comparable to those that occur in the testicular vein in situ, inhibited LH-stimulated testosterone secretion by rat testes perfused in vitro. This inhibition was rapid and dose responsive. In contrast to testosterone, intra-arterial infusion of 5 alpha-androstan-3 alpha, 17 beta-diol, corticosterone and androstenedione failed to inhibit testosterone secretion. The inhibition of testosterone secretion by intra-arterial infusion of testosterone was reversed by either cessation of testosterone infusion or the simultaneous addition of androstenedione to the artificial perfusion medium. Taken together, these results showed that LH-stimulated testosterone secretion by in vitro perfused rat testes was inhibited by a short-loop negative feedback of testosterone.

Measurement of testosterone with a high-performance liquid chromatograph equipped with a flow-through ultraviolet spectrophotometer.

A technique is described for measuring nanogram amounts of testosterone using high-performance liquid chromatography with detection by a flow-through spectrophotometer. The addition of a non-radioisotopic internal standard (4-androsten-11 beta-ol-3,17-dione) to the biological specimen automatically corrects for testosterone losses due to extraction and non-quantitative sample injection into the high-performance liquid chromatograph. This new method, which can be performed by inexperienced personnel, is shown to be rapid, precise, accurate and specific for testosterone.