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This work is concerned with the transmissibility of a disease, on observation of an outbreak of limited size. When such an outbreak occurs, an accurate estimate of the transmissibility of the responsible pathogen is essential for an appropriate response to future outbreaks. Transmissibility is usually characterised in terms of the reproduction number, R, which is the mean number of new cases of infection produced by a single infectious individual. A subcritical reproduction number (R < 1) guarantees that an outbreak will eventually die out of its own accord. By contrast, a supercritical reproduction number (R > 1) does not guarantee spread of the disease, since even with appreciable transmissibility, an outbreak may become extinct due to stochastic effects associated with a small number of infected individuals. Once the number of infectious individuals is conditioned on extinction, we show that an exact symmetry of the underlying theory ensures two distinct values of R, one larger than unity, the other smaller than unity, for which all outbreak properties are identical, with no signature of difference. Therefore a disease with a subcritical R, or its supercritical counterpart, when conditioned on extinction, have, for a given outbreak, identical individual likelihoods. In the full likelihood, this symmetry is lost, since the individual likelihood for a subcritical R is weighted by an extinction probability of unity, but the individual likelihood of a supercritical R is weighted by a sub-unity extinction probability. However, the inference can still benefit from the underlying symmetry, since it yields a mapping of all supercritical reproduction numbers onto the subcritical domain (R < 1), thereby speeding up evaluation of the likelihood profile. The symmetry holds in the standard situation, where the distribution of secondary cases is Poisson, as well as where this distribution has a negative binomial form and super-spreading can occur., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Adaptation is a defining property of living systems. It occurs when organisms become better suited to their environment. The phenomena that people find most fascinating about biological systems are, in general, the result of adaptive processes. Examples include the mammalian central nervous system, the flight of birds and insects, photosynthesis, and the human hand. However, despite the centrality of adaptation for biology, there is no generally agreed quantitative way to describe the degree to which an organism is adapted. Here, we address this situation by proposing a quantitative measure of adaptation. We also present results of computer simulations which demonstrate that some changes in parameter values cause mean adaptedness and mean relative fitness to change in opposite directions. This indicates that adaptedness and relative fitness are distinct concepts. We suggest that the measure of adaptedness proposed in this work may help to resolve questions about 'units of selection' and 'major transitions in evolution'., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this work we consider fixation of an allele in a population. Fixation is key to understanding the way long-term evolutionary change occurs at the gene and molecular levels. Two basic aspects of fixation are: (i) the chance it occurs and (ii) the way the gene frequency progresses to fixation. We present exact results for both aspects of fixation for the Wright-Fisher model. We give the exact fixation probability for some different schemes of frequency-dependent selection. We also give the corresponding exact stochastic difference equation that generates frequency trajectories which ultimately fix. Exactness of the results means selection need not be weak. There are possible applications of this work to data analysis, modelling, and tests of approximations. The methodology employed illustrates that knowledge of the fixation probability, for all initial frequencies, fully characterises the dynamics of the Wright-Fisher model. The stochastic equations for fixing trajectories allow insight into the way fixation occurs. They provide the alternative picture that fixation is driven by the injection of one carrier of the fixing allele into the population each generation. The stochastic equations allow explicit calculation of some properties of fixing trajectories and their efficient simulation. The results are illustrated and tested with simulations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Random processes in biology, in particular random genetic drift, often make it difficult to predict the fate of a particular mutation in a population. Using principles of theoretical population genetics, we present a form of biological control that ensures a focal allele's frequency, at a given locus, achieves a prescribed probability distribution at a given time. This control is in the form of an additional evolutionary force that acts on a population. We provide the mathematical framework that determines the additional force. Our analysis indicates that generally the additional force depends on the frequency of the focal allele, and it may also depend on the time. We argue that translating this additional force into an externally controlled process, which has the possibility of being implemented in a number of different ways corresponding to selection, migration, mutation, or a combination of these, may provide a flexible instrument for targeted change of traits of interest in natural populations. This framework may be applied, or used as an informed form of guidance, in a variety of different biological scenarios including: yield and pesticide optimisation in crop production, biofermentation, the local regulation of human-associated natural populations, such as parasitic animals, or bacterial communities in hospitals., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this work we assume that we have some knowledge about the state of a population at two known times, when the dynamics is governed by a Markov chain such as a Wright-Fisher model. Such knowledge could be obtained, for example, from observations made on ancient and contemporary DNA, or during laboratory experiments involving long term evolution. A natural assumption is that the behaviour of the population, between observations, is related to (or constrained by) what was actually observed. The present work shows that this assumption has limited validity. When the time interval between observations is larger than a characteristic value, which is a property of the population under consideration, there is a range of intermediate times where the behaviour of the population has reduced or no dependence on what was observed and an equilibrium-like distribution applies. Thus, for example, if the frequency of an allele is observed at two different times, then for a large enough time interval between observations, the population has reduced or no dependence on the two observed frequencies for a range of intermediate times. Given observations of a population at two times, we provide a general theoretical analysis of the behaviour of the population at all intermediate times, and determine an expression for the characteristic time interval, beyond which the observations do not constrain the population's behaviour over a range of intermediate times. The findings of this work relate to what can be meaningfully inferred about a population at intermediate times, given knowledge of terminal states., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Wright-Fisher model is an important model in evolutionary biology and population genetics. It has been applied in numerous analyses of finite populations with discrete generations. It is recognised that real populations can behave, in some key aspects, as though their size that is not the census size, N, but rather a smaller size, namely the effective population size, Ne. However, in the Wright-Fisher model, there is no distinction between the effective and census population sizes. Equivalently, we can say that in this model, Ne coincides with N. The Wright-Fisher model therefore lacks an important aspect of biological realism. Here, we present a method that allows Ne to be directly incorporated into the Wright-Fisher model. The modified model involves matrices whose size is determined by Ne. Thus apart from increased biological realism, the modified model also has reduced computational complexity, particularly so when Ne⪡N. For complex problems, it may be hard or impossible to numerically analyse the most commonly-used approximation of the Wright-Fisher model that incorporates Ne, namely the diffusion approximation. An alternative approach is simulation. However, the simulations need to be sufficiently detailed that they yield an effective size that is different to the census size. Simulations may also be time consuming and have attendant statistical errors. The method presented in this work may then be the only alternative to simulations, when Ne differs from N. We illustrate the straightforward application of the method to some problems involving allele fixation and the determination of the equilibrium site frequency spectrum. We then apply the method to the problem of fixation when three alleles are segregating in a population. This latter problem is significantly more complex than a two allele problem and since the diffusion equation cannot be numerically solved, the only other way Ne can be incorporated into the analysis is by simulation. We have achieved good accuracy in all cases considered. In summary, the present work extends the realism and tractability of an important model of evolutionary biology and population genetics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We consider the simplest form of negative frequency-dependent selection in a biallelic haploid population, where the selection coefficient of a mutant allele is a linear function of the allele's frequency, and changes from positive to negative as the frequency is increased. In an effectively infinite population this behaviour leads to a stable polymorphism. We present a theoretical investigation of what occurs in a finite population, where a long-lived polymorphism may be formed, but which fluctuates and ultimately disappears due to random genetic drift. We model the dynamics as a branching process and explicitly take into account differences between the census population size and the effective population size, which play different roles in the dynamics. We characterise the behaviour of the population in terms of three distinct timescales associated with: (i) early loss of mutant alleles, (ii) achievement of the long-lived polymorphism, (iii) disappearance of the polymorphism. Timescales (i) and (iii) depend on the effective population size and are, as a consequence, affected by random genetic drift, while timescale (ii) depends primarily on the census size and is relatively insensitive to genetic drift. Analysis and simulations of the branching process clarify the different influences of the census and effective population sizes. One substantial quantitative difference, between populations where the effective and census population sizes coincide and where they differ, lies in the number of mutant alleles in the long-lived polymorphism. This number is approximately proportional to the census size. Thus assuming the census size equals a much smaller effective population size predicts a much smaller number of mutants in the long-lived polymorphism., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We consider a population whose size changes stochastically under a branching process, with the added modification that each generation a fixed number of individuals are removed, irrespective of the size of the population. We call removal that is independent of population size 'hard harvesting'. A key feature of hard harvesting occurs if the size of the population is smaller than the fixed number that are harvested. In such a case, the dynamics cannot continue and must terminate. We find that even for populations with a tendency to grow, there is a finite probability of termination. We determine the probability of termination, and given that termination occurs, we characterise the statistical properties of the random time to termination. We determine the impact of hard harvesting on the size of the population, in populations where termination has not occurred., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The way population size, population structure (with migration), and spatially dependent selection (where there is no globally optimal allele), combine to affect the substitution rate is poorly understood. Here, we consider a two patch model where mutant alleles are beneficial in one patch and deleterious in the other patch. We assume that the spatial average of selection on mutant alleles is zero. We take each patch to maintain a finite number of N adults each generation, hence random genetic drift can independently occur in each patch. We show that the principal way the population size, N, when large, affects the substitution rate, R∞, is through its dependence on two composite parameters. These are the scaled migration rate M (∝ population size × migration rate), and the scaled selection intensity S (∝population size × beneficial effect of a mutant). Any relation between S and M that arises for ecological/evolutionary reasons can strongly influence the way the substitution rate, R∞, depends on the population size, N. In the simplest situation, both M and S are proportional to N, and this is shown to lead to R∞ increasing with N when S is not large. The behaviour, that R∞ increases with N, is not inevitable; a more complex relation between S and M can lead to the opposite or other behaviours. In particular, let us assume that dM/dN is positive, as would occur if the migration rate were constant, S is not large, and S depends on M (i.e., S=S(M)). We then find that if S(M) satisfies S(M)>((1+M)/1+2M)S(0) then the substitution rate, R∞, increases with N, but if S(M)<((1+M)/1+2M)S(0) then R∞ decreases with N., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Forward and backward simulations play an increasing role in population genetics, in particular when inferring the relative importance of evolutionary forces. It is therefore important to develop fast and accurate simulation methods for general population genetics models. Here we present an exact simulation method that generates trajectories of an allele׳s frequency in a finite population, as described by a general Wright-Fisher model. The method generates conditioned trajectories that start from a known frequency at a known time, and which achieve a specific final frequency at a known final time. The simulation method applies irrespective of the smallness of the probability of the transition between the initial and final states, because it is not based on rejection of trajectories. We illustrate the method on several different populations where a Wright-Fisher model (or related) applies, namely (i) a locus with 2 alleles, that is subject to selection and mutation; (ii) a locus with 3 alleles, that is subject to selection; (iii) a locus in a metapopulation consisting of two subpopulations of finite size, that are subject to selection and migration. The simulation method allows the generation of conditioned trajectories that can be used for the purposes of visualisation, the estimation of summary statistics, and the development/testing of new inferential methods. The simulated trajectories provide a very simple approach to estimating quantities that cannot easily be expressed in terms of the transition matrix, and can be applied to finite Markov chains other than the Wright-Fisher model., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A result is derived, in the form of a sum, for the time-dependent probability of fixation of an unlinked neutral locus. The result captures many of the key features of the probability of fixation in a highly compact form. For 'small' times (t ≲ 4N(e)) a single term of the sum accurately determines the time-dependent probability of fixation. This is in contrast to the well-known result of Kimura, which requires the contribution of many terms in a different sum, for 'small' times. Going beyond small times, an approximation is derived for the time-dependent probability of fixation which applies for all times when the initial relative allele frequency is small., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We investigate the detailed connection between the Wright-Fisher model of random genetic drift and the diffusion approximation, under the assumption that selection and drift are weak and so cause small changes over a single generation. A representation of the mathematics underlying the Wright-Fisher model is introduced which allows the connection to be made with the corresponding mathematics underlying the diffusion approximation. Two 'hybrid' models are also introduced which lie 'between' the Wright-Fisher model and the diffusion approximation. In model 1 the relative allele frequency takes discrete values while time is continuous; in model 2 time is discrete and relative allele frequency is continuous. While both hybrid models appear to have a similar status and the same level of plausibility, the different nature of time and frequency in the two models leads to significant mathematical differences. Model 2 is mathematically inconsistent and has to be ruled out as being meaningful. Model 1 is used to clarify the content of Kimura's solution of the diffusion equation, which is shown to have the natural interpretation as describing only those populations where alleles are segregating. By contrast the Wright-Fisher model and the solution of the diffusion equation of McKane and Waxman cover populations of all categories, namely populations where alleles segregate, are lost, or fix., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

On the basis of experimental observations, this paper develops two well-defined mathematical models for the level of activity of Pharaoh's ants within their nesting area, with the aim of providing a more general understanding of animal activity. Under specific conditions, we observe that the activity of ants within their nesting area appears to show no dependence on their density. Making the assumption that all ants move independently of one another, this behaviour can be mathematically modelled as a random process based on the binomial distribution. Developing the model on this basis allows an exponential distribution to be exposed that underlies the time-intervals between ants leaving the nesting area. Such a distribution is present, irrespective of whether the ant population in the nesting area remains constant or steadily depletes, and suggests that ant-ant interactions do not play any significant role in determining ant activity under the experimental conditions adopted. The mathematical framework presented plays the role of a null model that will have a wide range of applications for detecting other determinants of activity-level (not addressed in this study) including environmental and social factors such as food availability, temperature, humidity, presence of pheromone trails, along with intraspecific and interspecific interactions outside the nest and, indeed, more generally. The null model should have applications to a range of organisms. Lastly, we discuss our data in relation to a recent study of ants leaving their nest (Richardson et al., 2010) in which the null model was rejected in favour of record dynamics, where ant-ant interactions were conjectured to play a role., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This work presents a new approach to Muller's ratchet, where Haigh's model is approximately mapped into a simpler model that describes the behaviour of a population after a click of the ratchet, i.e., after loss of what was the fittest class. This new model predicts the distribution of times to the next click of the ratchet and is equivalent to a Wright-Fisher model for a population of haploid asexual individuals with one locus and two alleles. Within this model, the fittest members of a population correspond to carriers of one allele, while all other individuals have suboptimal fitness and are represented as carriers of the other allele. In this way, all suboptimal fitness individuals are amalgamated into a single "mutant" class. The approach presented here has some limitations and the potential for improvement. However, it does lead to results for the rate of the ratchet that, over a wide range of parameters, are accurate within one order of magnitude of simulation results. This contrasts with existing approaches, which are designed for only one or other of the two different parameter regimes known for the ratchet and are more accurate only in the parameter regime they were designed for. Numerical results are presented for the mean time between clicks of the ratchet for (i) the Wright-Fisher model, (ii) a diffusion approximation of this model and (iii) individually based simulations of a full model. The diffusion approximation is validated over a wide range of parameters by its close agreement with the Wright-Fisher model. The present work predicts that: (a) the time between clicks of the ratchet is insensitive to the value of the selection coefficient when the genomic mutation rate is large compared with the selection coefficient against a deleterious mutation, (b) the time interval between clicks of the ratchet has, approximately, an exponential distribution (or its discrete analogue). It is thus possible to determine the variance in times between clicks, given the expected time between clicks. Evidence for both (a) and (b) is seen in simulations., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Retrotransposons occur in extremely large numbers in many eukaryotic genomes. However, little is known of the factors which affect the distribution of close proximity elements. In this work we investigate the frequency of close facing retrotransposons in a plant species with extremely high numbers of retrotransposons. Molecular observations are compared with predictions of a mathematical model that assumes a uniform probability of retrotransposon insertion into the genome. The mathematical model plays the role of a null hypothesis. We find that compared with the predictions of the model, there is a statistically significant deficit of identical copies of facing retroelements that are close to one another. This suggests that an efficient mechanism exists that removes or limits close facing retroelements., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

The development and implementation of programs in the U.S. to minimize risks and assess unintended consequences of new medications has been increasingly required by the Food and Drug Administration (FDA) since the mid 1990s. This paper provides four case histories of risk management and post-marketing surveillance programs utilized recently to address problems associated with possible abuse, dependence and diversion. The pharmaceutical sponsors of each of these drugs were invited to present their programs and followed a similar template for their summaries that are included in this article. The drugs and presenting companies were OxyContin, an analgesic marketed by Purdue Pharma L.P., Daytrana and Vyvanse, ADHD medications marketed by Shire Pharmaceuticals, Xyrem for narcolepsy marketed by Jazz Pharmaceuticals, and Subutex and Suboxone for opioid dependence marketed by Reckitt Benckiser Pharmaceuticals Inc. These case histories and subsequent discussions provide invaluable real-world examples and illustrate both the promise of risk management programs in providing a path to market and/or for keeping on the market drugs with serious potential risks. They also illustrate the limitations of such programs in actually controlling unintended consequences, as well as the challenge of finding the right balance of reducing risks without posing undue barriers to patient access. These experiences are highly relevant as the FDA increasingly requires pharmaceutical sponsors to develop and implement the more formalized and enforceable versions of the risk management term Risk Evaluation and Mitigation Strategies (REMS).

We consider the Wright Fisher model for a finite population of diploid sexual organisms where selection acts at a locus with multiple alleles. The mathematical description of a such a model requires vectors and matrices of a multidimensional nature, and hence has a considerable level of complexity. In the present work we avoid this complexity by introducing a simple mathematical transformation. This yields a description of the model in terms of ordinary vectors and ordinary matrices, thereby allowing standard linear algebra techniques to be directly employed. The new description yields a common mathematical representation of the Wright Fisher model that applies for arbitrary numbers of alleles. Within this framework, it is shown how the dynamics decomposes into component parts that are responsible for the different possible transitions of segregating and fixed populations, thereby allowing a clearer understanding of the population dynamics. This decomposition allows expressions to be directly derived for the mean time of fixation, the mean time of segregation (i.e., the sojourn time) and the probability of fixation. Numerical methods are discussed for the evaluation of these quantities.

We investigate a genetic model of a large population of sexual organisms in a changing environment. The organisms are subject to stabilising selection on a quantitative trait, with environmental change causing the fitness optimum to move. When the fitness optimum moves slowly, adaptation to the changing environment occurs by means of reasonably well-separated substitutions at the loci controlling the trait. In this way, the trait generally tracks the moving optimum, but in such a case, the population may exhibit periods of time where the mean trait value overshoots the moving optimal trait value, thereby exhibiting an apparent anticipation of selection. The mechanism underlying this phenomenon is determined from consideration of a simpler model that correctly captures the observed dynamical behaviour. We note that very slow rates of changes of traits are seen in the fossil record and the present work may be relevant to this topic.

Phylogenetic comparative methods have been used to study patterns of correlated evolution between biological traits of all kinds, and are increasingly used to identify predictors of the rate of DNA substitution. Substitution rate differs from most traits studied in that it cannot be observed directly, but must be inferred from substitutions accrued over a long period of time. Studying a mathematical model of trait and rate evolution, we show that the special nature of substitution rates can lead to a severe loss of power for standard comparative methods. We further show how strategies designed to maximise power, by increasing the number of data points, can have the opposite effect when substitution rate is involved. We then propose two modifications of the standard methods that can mitigate these problems. First, we show how pre-existing tests for homogeneity of variance can be used to identify and exclude those data from which changes in substitution rate cannot be reliably inferred. Second, we show that power can be increased by comparing substitution rate with the time average of the predictor trait along the history of the lineage, and introduce a maximum likelihood estimator of this quantity.

The forward diffusion equation for gene frequency dynamics is solved subject to the condition that the total probability is conserved at all times. This can lead to solutions developing singular spikes (Dirac delta functions) at the gene frequencies 0 and 1. When such spikes appear in solutions they signal gene loss or gene fixation, with the "weight" associated with the spikes corresponding to the probability of loss or fixation. The forward diffusion equation is thus solved for all gene frequencies, namely the absorbing frequencies of 0 and 1 along with the continuous range of gene frequencies on the interval (0,1) that excludes the frequencies of 0 and 1. Previously, the probabilities of the absorbing frequencies of 0 and 1 were found by appeal to the backward diffusion equation, while those in the continuous range (0,1) were found from the forward diffusion equation. Our unified approach does not require two separate equations for a complete dynamical treatment of all gene frequencies within a diffusion approximation framework. For cases involving mutation, migration and selection, it is shown that a property of the deterministic part of gene frequency dynamics determines when fixation and loss can occur. It is also shown how solution of the forward equation, at long times, leads to the standard result for the fixation probability.

Fisher's geometrical model of evolutionary adaptation has recently been used in a variety of contexts of interest to evolutionary biologists. The renewed interest in this model strongly motivates generalizations that make it a more realistic description of evolutionary adaptation. Previously, the distribution of mutant effects has, for analytical tractability, rather than biological realism, been taken as spherically symmetric. Here we substantially extend Fisher's model, by allowing a wider class of mutational distributions that incorporate mutational bias and more general deviations from spherical symmetry such as correlations between mutant effects. We also incorporate work on generalized fitness landscapes, thereby reducing the number of artificial assumptions underlying the model. The generalized model exhibits a substantially increased flexibility and a far richer underlying geometry. We find that the distribution characterizing selection coefficients of new mutations is expressed in terms of a number of geometrical invariants associated with mutation, selection and the parental phenotype.

We investigate a model that describes the evolution of a diploid sexual population in a changing environment. Individuals have discrete generations and are subject to selection on the phenotypic value of a quantitative trait, which is controlled by a finite number of bialleic loci. Environmental change is taken to lead to a uniformly changing optimal phenotypic value. The population continually adapts to the changing environment, by allelic substitution, at the loci controlling the trait. We investigate the detailed interrelation between the process of allelic substitution and the adaptation and variation of the population, via infinite population calculations and finite population simulations. We find a simple relation between the substitution rate and the rate of change of the optimal phenotypic value.

The nk model of fitness interactions is examined. This model has been used by previous authors to investigate the effects of fitness epistasis on substitution dynamics in molecular evolution, and to make broader claims about the importance of epistasis. To examine these claims, an infinite-allele approximation is introduced. In this limit, it is shown that the nk model is, at an appropriate level of description, formally identical to the non-epistatic House-of-Cards model--a well-studied model in theoretical population genetics. It is further shown that in many parameter regimes, the analytical results obtained from this infinite-allele approximation are very close to results from the full nk model (with a finite number of alleles per locus). The findings presented shed light on a number of previous results.

Background: Rats treated chronically in a large, open field with the dopamine D2/D3 receptor agonist quinpirole (QNP) develop compulsive checking behavior as defined by a set of behavioral criteria. This paradigm has been suggested as an animal model of obsessive-compulsive disorder (OCD). Because nicotine blocks various behaviors induced by ontogenetic QNP administration, we asked whether nicotine could attenuate QNP-induced compulsive checking., Methods: Adult male Long-Evans rats (n = 14/group) were treated twice weekly with saline (control), or with QNP (0.5 mg/kg) for 14-16 injections. On the last two injections, rats were pretreated in random order with an acute dose of nicotine (0.3 mg/kg base) or saline 10 min before administration of QNP or saline; and the effects on checking behavior was examined. The effects of chronic QNP treatment on nicotinic receptors in discrete brain regions were also determined., Results: Chronic QNP resulted in compulsive checking and increases in cerebellar alpha4beta2 and alpha7 nicotinic receptor densities. Nicotine pretreatment significantly reduced one of the three measures of compulsive checking behavior., Conclusions: Nicotine attenuates some symptoms of compulsive checking in a rat model of OCD; however, the mechanisms of this effect and therapeutic efficacy of nicotinic agonists in OCD require further study.

The intracellular signalling molecule and transcriptional activator STAT5b is a key mediator of the effects of intermittent plasma growth hormone (GH) pulses on the male-specific pattern of liver gene expression and pubertal body growth rates in rodents. Experiments with Stat5b gene-knockout mice have revealed that these GH-regulated, male-specific phenotypes are a direct consequence of GH pulse-dependent STAT5b activation and that loss of function of STAT5b cannot be compensated for by the closely related signalling molecule STAT5a. Physiological plasma GH pulses are required to obtain the high levels of activated STAT5b seen in the livers of males, and down-regulation of the GH receptor (GHR)-JAK-STAT5b pathway in hepatocytes exposed to GH in a near-continuous fashion underlies the low level of liver STAT5b activity that is characteristic of adult female rats. Termination of nuclear STAT5b signalling occurs at the conclusion of a plasma GH pulse, with STAT5b deactivation catalysed by a tyrosine phosphatase. In males, termination of the intracellular signalling stimulated by a plasma GH pulse is proposed to be additionally facilitated by GH-STAT5b-inducible SOCS-CIS proteins, which block the further activation of STAT5b by binding to and inhibiting the action of the GHR-JAK2 complex via multiple mechanisms. In this manner, the liver cell is rendered temporarily unresponsive to further GH-signalling events. SOCS-CIS proteins synthesized in liver cells stimulated continuously with GH may also contribute to the apparent down-regulation of STAT5b signalling that is observed in the female rat liver.

The biochemistry of foreign compound metabolism and the roles played by individual cytochrome P450 (CYP) enzymes in drug metabolism and in the toxification and detoxification of xenochemicals prevalent in the environment are important areas of molecular pharmacology and toxicology that have been widely studied over the past decade. Important advances in our understanding of the mechanisms through which foreign chemicals impact on these P450-dependent metabolic processes have been made during the past 2 years with several key discoveries relating to the mechanisms through which xenochemicals induce the expression of hepatic P450 enzymes. Roles for three "orphan" nuclear receptor superfamily members, designated CAR, PXR, and PPAR, in respectively mediating the induction of hepatic P450s belonging to families CYP2, CYP3, and CYP4 in response to the prototypical inducers phenobarbital (CAR), pregnenolone 16alpha-carbonitrile and rifampicin (PXR), and clofibric acid (PPAR) have now been established. Two other nuclear receptors, designated LXR and FXR, which are respectively activated by oxysterols and bile acids, also play a role in liver P450 expression, in this case regulation of P450 cholesterol 7alpha-hydroxylase, a key enzyme of bile acid biosynthesis. All five P450-regulatory nuclear receptors belong to the same nuclear receptor gene family (family NR1), share a common heterodimerization partner, retinoid X-receptor (RXR), and are subject to cross-talk interactions with other nuclear receptors and with a broad range of other intracellular signaling pathways, including those activated by certain cytokines and growth factors. Endogenous ligands of each of those nuclear receptors have been identified and physiological receptor functions are emerging, leading to the proposal that these receptors may primarily serve to modulate hepatic P450 activity in response to endogenous dietary or hormonal stimuli. Accordingly, P450 induction by xenobiotics may in some cases lead to a perturbation of endogenous regulatory circuits with associated pathophysiological consequences., (Copyright 1999 Academic Press.)

Chloroacetaldehyde (CA) is a nephrotoxic and neurotoxic metabolite of the anticancer drug ifosfamide (IFA) and is a dose-limiting factor in IFA-based chemotherapy. Plasma levels of CA in IFA-treated cancer patients are often difficult to determine due to the lack of a sufficiently sensitive and specific analytical method. We have developed a simple and sensitive HPLC method with fluorescence detection to measure CA formation catalyzed by liver cytochrome P450 enzymes, either in vivo in IFA-injected rats or in vitro in liver microsomal incubations. This method is based on the formation of the highly fluorescent adduct 1-N(6)-ethenoadenosine from the reaction of CA with adenosine (10 mM) at pH 4.5 upon heating at 80 degrees C for 2 h. The derivatization mixture is directly injected onto a C18 HPLC column and is monitored with a fluorescence detector. Calibration curves are linear (r > 0.999) over a wide range of CA concentrations (5-400 pmol). The limit of detection of CA in plasma using this method is <0.1 microM and only 50 microl of plasma is required for the assay. By coupling this method with a recently described HPLC-fluorescent method to determine acrolein, a cytochrome P450 metabolite of IFA formed during the activation of the drug by 4-hydroxylation, the two major, alternative P450-catalyzed pathways of IFA metabolism can be monitored from the same plasma samples or liver microsomal incubations and the partitioning of drug between these two pathways thereby quantitated. This assay may prove to be useful for studies of IFA metabolism aimed at identifying factors that contribute to individual differences in CA formation and in developing approaches to minimize CA formation while maximizing IFA cytotoxicity., (Copyright 1999 Academic Press.)

The membrane-bound endogenous fatty acid arachidonic acid can be released from membranes by phospholipases and then metabolized to biologically active compounds by cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) enzymes. In the liver the CYP pathway is the most significant. Liver CYP arachidonate products include epoxyeicosatrienoic acids (EETs) and monohydroxylated products (HETEs). We examined metabolism of [1-14C]arachidonic acid by a panel of 10 human CYP enzymes expressed in HepG2 cells. In the absence of expressed CYP enzymes, control HepG2 cell microsomes generated only small amounts of omega- and omega--1-OH arachidonic acid (ratio 2:1). Microsomes from HepG2 cells expressing CYP2C8, 2C9, 1A2, and 2E1 were 7-21 times more active than microsomes from the HepG2 controls. CYP2C8, 2C9, and 1A2 principally generated epoxygenase products; 36 to 48% were in the form of EET-diols, reflecting host HepG2 microsomal epoxide hydrolase activity. CYP2C8 and 2C9 formed more 14,15- and 11,12-EET than did CYP1A2, while CYP1A2 formed more 8,9-EET. CYP2C9 also generated a peak with the retention time of 12-HETE. CYP2E1 generated omega--1-OH arachidonic acid and, to a lesser extent, omega-OH arachidonic acid (ratio 2:1). A small amount of epoxygenase activity was also detected for CYP2B6; its overall activity, however, was only about twice control levels. Activities of CYP2A6, 3A3, 3A4, and 3A5 were low and limited to the omega-/omega--1-OH arachidonic acid peak; CYP2D6 was inactive. Microsomes prepared from three individual human livers varied threefold in total arachidonic acid metabolism. For all three livers omega-OH arachidonic acid was the major product (up to 74% of total metabolites). Epoxygenase products constituted 14 to 28% of the total products; 60 to 83% of those were EET-diols, indicating that the human liver microsomes have substantial EET-epoxide hydrolase activity. 11,12-EET was the major EET for two livers and 14,15-EET for the third. The CYP2C inhibitor sulfaphenazole depressed human liver microsomal epoxygenase activity by 50% at 50 microM, while alpha-naphthoflavone inhibited arachidonic acid epoxygenase activity by 27% at 2 microM and by 32% at 10 microM. Collectively, these findings suggest that human liver microsomal arachidonic acid metabolism is catalyzed principally by CYP2C enzymes. CYP1A2, CYP2E1, and possibly CYP2B6 are likely to play more minor roles, though their contribution may be enhanced by exposure to inducers of those enzymes. CYP2A6, CYP2D6, and CYP3A enzymes are unlikely to make any significant contribution.(ABSTRACT TRUNCATED AT 400 WORDS)

Sexually dimorphic regulation of phenobarbital-induced cytochromes P450 2B1 and 2B2 (collectively referred to as P450 2B) as well as P450 2B-dependent monooxygenase activities was studied in multi-hormone-depleted hypophysectomized rats and in growth hormone (GH)-deficient monosodium glutamate (MSG)-treated rats. Our results indicate that endogenous GH suppresses phenobarbital induction of P450 2B and that the feminine pattern of continuous GH secretion is more suppressive than the masculine profile of episodic secretion. Moreover, we have found that it is the height of the GH pulse, and not necessarily its frequency nor the interpulse trough periods, that signals the suppressive effects of GH on P450 2B expression in male rats. Last, irrespective of the presence or absence of circulating GH, the magnitude of phenobarbital induction of P450 2B and associated monooxygenases was consistently lower in female rats, suggesting the presence of some degree of sex-dependent, but GH-independent regulation.

A variety of chemicals, including triacetyloleandomycin (TAO), alpha-naphthoflavone (ANF), and diethyldithiocarbamate (DDC), are widely used as inhibitory probes for select individual human cytochrome P450 (CYP) enzymes, despite the fact that the selectivity of these inhibitors has not been rigorously evaluated. In the present study we take advantage of recent advances in cDNA-directed human P450 expression to evaluate directly the P450 form selectivity of TAO, ANF, and DDC, using a panel of 10 individual cDNA-expressed human P450s. Under experimental conditions known to yield maximal TAO complexation with P450 hemoproteins, TAO (20 microM) inhibited the catalytic activity of expressed CYPs 3A3, 3A4, and 3A5, whereas it did not affect CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, or 2E1 activity. ANF inhibited not only CYPs 1A1 and 1A2 (IC50 = 0.4-0.5 microM), but it was also similarly effective against CYPs 2C8 and 2C9. Increasing the concentration of ANF to 10 microM led to inhibition of CYP2A6 and CYP2B6. Although a previous study suggested that DDC is a selective inhibitor of CYP2E1, the present investigation shows that at concentrations required to inhibit CYP2E1 (IC50 approximately 125 microM when preincubated with NADPH), DDC also inhibited CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, and 3A4. Decreasing the concentration of DDC to 10 microM, however, led to inhibition of CYP2A6 (65% inhibition) and CYP2B6 (50% inhibition), but none of the other P450s examined, including CYP2E1. Overall, these results establish that (a) TAO is a selective inhibitor of the human CYP3A subfamily; (b) ANF potently inhibits CYP2C8 and CYP2C9, in addition to CYPs 1A1 and 1A2; and (c) DDC cannot be employed as a diagnostic inhibitory probe for CYP2E1.

The alkylating agent BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] can be inactivated through denitrosation reactions catalyzed by both cytosolic and microsomal enzymes. While previous studies have identified a class mu glutathione S-transferase [rat transferase 4-4 (Yb2)] as a major catalyst of the cytosolic denitrosation reaction, the enzymatic catalysts of BCNU denitrosation in microsomal membranes have not been identified. In the present study, both NADPH and glutathione (GSH) were found to support BCNU denitrosation catalyzed by isolated rat liver microsomes. Treatment of rats with the microsomal enzyme inducers phenobarbital and dexamethasone increased NADPH-dependent liver microsomal BCNU denitrosation up to fivefold without major effect on the GSH-dependent denitrosation activity. Although the NADPH-dependent activity was fully inhibited by antibody to NADPH-P450 reductase, purified NADPH-P450 reductase catalyzed BCNU denitrosation at rates that could only account for approximately 2-3% of the microsomal activity. Other experiments, including selective inhibition of NADPH-dependent microsomal BCNU denitrosation by chemical and antibody inhibitors of cytochrome P450, competitive inhibition of P450-catalyzed cyclophosphamide and ifosfamide activation by BCNU, and reconstitution of the denitrosation reaction by purified P450 enzyme 2B1 (major phenobarbital-inducible P450 form), established an important role for cytochrome P450 in BCNU denitrosation. By contrast, GSH-dependent microsomal BCNU denitrosation was unaffected by cytochrome P450 inhibitors, but was inhibited, with varying degrees of selectivity, by the microsomal glutathione S-transferase inhibitors ethacrynic acid, bromosulfophthalein, and indomethacin. These studies establish that BCNU inactivation can be catalyzed by two independent microsomal enzyme systems and suggest that therapeutically useful improvements in BCNU antitumor activity might be achieved through differential inhibition of these enzyme systems in tumor as compared to extratumoral sites.

Suicide inactivation of hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1/A2 by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) in intact rats results in prosthetic heme destruction, albeit by apparently distinct mechanisms. Such heme destruction is now shown to be associated with the loss of immunochemically detectable apoprotein of P450s 2C11 and 3A but with little of that of P450 2C6, in spite of their comparable DDEP-mediated functional inactivation. The loss of a approximately 50-kDa hepatic microsomal protein band along with the immunoreactive P450 3A loss strengthens the concept that such an in vivo loss indeed reflects proteolysis of the DDEP-inactivated P450. Furthermore, this propensity of DDEP-inactivated P450s 3A for proteolysis appears to correlate with the relative degree of prosthetic heme alkylation of their apoprotein rather than their functional inactivation per se. Thus, rapid degradation of apoP450s 3A was seen after DDEP treatment, which promoted extensive irreversible heme binding to apoP450s 3A, but not after exposure to allylisopropylacetamide (AIA), which inactivates these isozymes comparably, but induced minimal apoP450 3A heme alkylation. In addition, differences were observed in the relative sensitivities of proteolysis of DDEP-inactivated P450s 2C11 and 3A to hemin, which largely prevented the DDEP-induced proteolytic loss of P450 2C11 but apparently failed to prevent the loss of DDEP-inactivated P450s 3A, when coadministered with DDEP. This differential hemin sensitivity of the proteolysis of DDEP-inactivated P450 2C11, coupled with the observation that immunochemically detectable P450 2C11 loss occurs after its inactivation by both AIA and DDEP, provides compelling support for the existence of distinct proteolytic pathways for individual suicidally inactivated P450s.

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.

Cytochrome P-450 cholesterol 7 alpha-hydroxylase (P-450Ch7 alpha) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids. Incubation of rat liver microsomes in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer resulted in a time-dependent deactivation of P-450Ch7 alpha which was markedly accelerated by the nonionic detergent Tween 80. Microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450-dependent 7-ethoxycoumarin O-deethylase activities were unaffected under these conditions, evidencing the selectivity of the deactivation process for P-450Ch7 alpha. The rate (t 1/2 = 15-19 min at 37 degrees C) and maximal extent of P-450Ch7 alpha deactivation (greater than or equal to 90%) were both unaffected by the presence of cytosolic proteins and were also not dependent on the initial enzyme level, as shown using liver microsomes isolated from untreated, cholestyramine-fed, and xenobiotic-induced rats exhibiting an eight-fold range in P-450Ch7 alpha activity. Scavengers for reduced oxygen species were also without effect. P-450Ch7 alpha was stabilized some six- to sevenfold (t 1/2 = 94-143 min) by the phosphatase inhibitor NaF. Of a series of other phosphatase inhibitors examined, including, among others, EDTA, vanadate, and molybdate, only phosphate-containing compounds and the calmodulin antagonist trifluoperazine, and inhibitor of the Ca2+-calmodulin-dependent phosphatase calcineurin, effectively stabilized P-450Ch7 alpha. Modulation of P-450Ch7 alpha deactivation by these inhibitors generally paralleled their effects on isolated calcineurin. A variety of structurally diverse calmodulin antagonists examined were also found to effectively protect P-450Ch7 alpha from deactivation; these include calmidazolium and tamoxifen (IC50 = 25 to 50 microM), chlorpromazine, thioridazine, amitriptyline, imipramine, and the naphthalene sulfonamide compound W-7 (IC50 = 50 to 300 microM). Structure-activity analysis of several phenothiazines and their derivatives indicated that although little activity was exhibited by the sulfoxides, some protection was provided by the corresponding sulfones. On the basis of these observations, various models for the molecular basis of enzyme deactivation are considered, including the hypothesis that a calcineurin-like microsomal phosphatase mediates deactivation of this cytochrome P-450 enzyme.

The hydroxylation of lithocholic acid (3 alpha-hydroxy-5 beta-cholanoic acid) by adult male Sprague-Dawley rat liver microsomes supplemented with NADPH was studied. Metabolites were separated by a combination of thin-layer chromatography and high pressure liquid chromatography, both with and without prior methylation and acetylation of the samples. The resulting products were characterized by thin-layer, gas-liquid, and high pressure liquid chromatography by comparison with authentic bile acid standards; final structure determination was by proton nuclear magnetic resonance spectroscopy and by mass spectrometry. The following reaction products were found: 3 alpha, 6 beta-dihydroxy-5 beta-cholanoic acid (80% of total metabolites) and 3 alpha, 6 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 7 alpha-dihydroxy-5 beta-cholanoic, 3 alpha, 6 beta,7 beta-trihydroxy-5 beta-cholanoic, and 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acids (less than or equal to 5% each). In addition, one unidentified trihydroxylic bile acid and several minor compounds were present. It is concluded that four different hydroxylation reactions of lithocholic acid, namely the predominant 6 beta as well as the minor 6 alpha, 7 alpha, and 7 beta hydroxylations, are catalyzed by rat hepatic microsomes; 7 beta-hydroxylation may occur only with dihydroxylated bile acids but not with lithocholate itself. The presence of the 6-oxo bile acid can be explained either by direct oxidation of a hydroxyl group by cytochrome P-450, or by the action of microsomal dehydrogenase(s) which could also catalyze the epimerization of hydroxyl groups via their oxidation. The results form the basis of a proposed scheme of the oxidative metabolism of lithocholic acid in rat liver microsomes.

Cytochrome P-450g was isolated from livers of adult male Sprague-Dawley (CD) rats. Antibody to P-450g cross-reacted with several proteins in Western blots of liver microsomes from male CD rats. An immunospecific antibody was prepared by adsorption over immunoaffinity columns of Sepharose-bound solubilized rat liver microsomes from female CD and male Fischer 344 rats containing little or no P-450g. The immunopurified antibody recognized a single protein on Western blots of liver microsomes from male CD rats with an electrophoretic mobility identical to that of P-450g. Using this antibody, P-450g was shown to be male specific in the CD rat and expressed at maturity. Adult male CD rats were shown to fall into two distinct populations, those expressing high levels of P-450g (+g) and those expressing low levels of P-450g (-g). The P-450g content of the two populations differed 10- to 20-fold. P-450g was low or absent in liver microsomes of both sexes of adult Fischer rats. Purified P-450g catalyzed the hydroxylation of testosterone and androstenedione principally at the 6 beta-position and progesterone at the 16 alpha- and 6 beta-positions in reconstituted systems. However, the hydroxylation of these steroids by liver microsomes from the (+g) phenotype did not differ from that of the (-g) phenotype. Translatable mRNA for P-450g could be detected in livers of adult male CD rats but not female rats. However, the level of P-450g mRNA in livers of adult male CD rats with the (+g) phenotype did not differ from that of (-g) phenotype. These data suggest that phenotypic differences in the expression of P-450g do not depend on differences in mRNA content. This study provides a clear example of a P-450 isozyme which is markedly variable in an outbred strain of rat and absent in an inbred strain. Such a marked variability in an enzyme involved in metabolism of endogenous and exogenous substrates could account for some of the strain differences in susceptibility to toxic chemicals.

Rat cytochrome P450 2c (P450 gene IIC11) is a constitutive, male-specific hepatic enzyme which is suppressed greater than 90% by treatment with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) [H. N. Yeowell et al. (1987) Mol. Pharmacol. 32, 340-347]. HCB also decreases serum testosterone levels in adult male rats (greater than 98% loss). The present study assesses whether the suppression of P450 2c by HCB is a direct result of its effects on serum testosterone levels. Further, the site along the hypothalamic-pituitary-testicular axis at which HCB acts to depress testosterone secretion was examined. Administration of the synthetic androgen methyltrienolone to HCB-treated rats failed to prevent the suppression of P450 2c mRNA and its associated microsomal steroid 16 alpha-hydroxylase activity under conditions where it effectively reversed the large decrease in P450 2c mRNA and steroid 16 alpha-hydroxylase activity produced by castration. Hepatic steroid 6 beta-hydroxylase activity, which is catalyzed primarily by P450 2a (P450 gene IIIA2), was also suppressed by HCB and was not protected by methyltrienolone. Administration of either human chorionic gonadotropin, an analog of pituitary-derived luteinizing hormone, or the hypothalamic luteinizing hormone releasing hormone elevated serum testosterone levels to a much smaller extent in HCB-treated rats than in control rats. These results indicate that the effects of HCB on serum testosterone levels reflect its effects on testicular function rather than the pituitary or hypothalamus. However, the present study demonstrates that the consequential reduction in serum testosterone levels in HCB-treated rats is not causally related to the reduction in hepatic P450 2c levels. Thus, HCB must also act on some other regulatory mechanism involved in the expression of this protein.

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.

Cytochrome P-450Ch7 alpha (cholesterol 7 alpha-hydroxylase) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids in mammalian liver. The properties of this cytochrome P-450 (P-450) form were studied in rat hepatic microsomal preparations in comparison to those of several well characterized constitutive and xenobiotic-inducible rat hepatic P-450s. Administration of the bile acid-sequestering resin cholestyramine [4% (w/w) in the diet] to male or female rats maintained on a reverse light cycle led to a 10- to 15-fold induction of P-450Ch7 alpha activity relative to untreated, standard light cycle controls. By contrast, the levels of four hepatic steroid hormone hydroxylating P-450 enzymes, designated 2a, 2c, 3, and PB-4 [Waxman, D.J. (1984) J. Biol. Chem. 259, 15481-15490], were not significantly affected by cholestyramine treatment. Antibody inhibition experiments established that P-450Ch7 alpha is immunochemically distinct from nine other rat hepatic P-450s, including P-450 3, a highly regio- and stereoselective steroid hormone 7 alpha-hydroxylase. P-450Ch7 alpha was shown to be selectively inactivated by micromolar concentrations of the disulfide-containing reagents disulfiram (Antabuse) and 2,2'-dithiopyridine. This inactivation was readily reversed upon incubation with 2-mercaptoethylamine, suggesting the presence of a highly reactive thiol group at the active site of P-450Ch7 alpha. These findings demonstrate that P-450Ch7 alpha corresponds to a unique P-450 enzyme exhibiting inductive, biochemical, immunochemical, and regulatory properties distinct from those of nine well-characterized rat hepatic P-450 forms.

NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.

Folylpolyglutamate synthetase (FPGS) was isolated from human liver cytosol by 0-30% (w/v) ammonium sulfate fractionation and characterized biochemically. Using aminopterin (AMT), L-[3H]glutamate and MgATP as cosubstrates, maximal gamma-L-glutamylation activity was observed in the presence of the activators KCl and NaHCO3. ATP and 2-mercaptoethanol were each required for enzyme activity and stability. In the absence of ATP, human liver FPGS rapidly inactivated at 37 degrees C (t1/2 approximately 8 min), whereas FPGS isolated from rabbit liver was significantly more stable (t1/2 = 68 min). Both folates and antifolates were effectively polyglutamylated by the isolated human liver enzyme. Km parameters determined for AMT (Km = 4.3 microM) were similar to those determined for several reduced folates (tetrahydrofolic acid, dihydrofolic acid, and folinic acid; Km = 3-7 microM), while significantly higher Km values were observed for methotrexate (MTX) and 5-methyltetrahydrofolic acid (Km = 50-60 microM) and for folic acid (Km = 100 microM). All of the substrates examined exhibited Vmax values ranging from 30 to 90% of the AMT value (Vmax = 935 pmol product/mg/h). The order of reactivity for these substrates differed from that determined in parallel studies for FPGS isolated from rat and rabbit liver. In the case of AMT and several reduced folates, inhibition of human liver FPGS was observed at substrate concentrations at or above 50-250 microM. FPGS isolated from six individual human livers exhibited highly similar biochemical and kinetic properties, suggesting the presence of the same or at least highly similar enzyme species in each individual, with a five-fold interindividual range in specific activities observed. Comparison of MTX with its higher polyglutamates (MTX-Glu2 to MTX-Glu6) as FPGS substrates indicated a significant decrease in Vmax values with increasing glutamate chain length which was partially compensated for by a corresponding decrease in Km. Consistent with these observations, the isolated enzyme was unable to synthesize polyglutamates higher than MTX-Glu3 when MTX was supplied as substrate, raising the question as to how MTX polyglutamates containing up to five or six gamma-L-glutamate residues are formed in vivo.

Preparations of hepatic cytochrome P-450 h [D. E. Ryan, et al. (1984) J. Biol. Chem. 259, 1239] and cytochrome P-450 2c [D. J. Waxman (1984) J. Biol. Chem. 259, 15481] from outbred Sprague-Dawley rats were analyzed using two-dimensional electrophoresis and in situ peptide mapping. Both preparations consisted of the same isozyme which was previously characterized as a developmentally regulated, male-specific cytochrome P-450 active in the 16 alpha-hydroxylation of steroids. Each preparation evidenced microheterogeneity which was shown, in part, to result from the existence of two genetically determined variant forms of cytochrome P-450 h/2c. Analyses of hepatic microsomes from several inbred strains of rat revealed that each was characterized by a single variant form of this isozyme, with some strains expressing a variant that was not present in Sprague-Dawley rats. Genetic crosses indicated that these electrophoretic variants represent allozymic forms of cytochrome P-450 h/2c which are codominantly expressed at a single autosomal locus. Additional microheterogeneity of each allozymic form of cytochrome P-450 h/2c was shown to result from a specific in vitro modification that may involve limited proteolysis near its C terminus by a microsome-bound protease.