An association of intrapartum synthetic oxytocin dosing and the odds of developing autism.

LAY ABSTRACT: Oxytocin is a hormone naturally produced in the human body that can make the womb (uterus) contract during labor. Manufactured oxytocin is frequently given to mothers in labor to strengthen the contractions or in some cases to start labor. This study compared children with a diagnosis of autism and children without autism to see whether children with autism received more oxytocin during labor. The odds of a child having an autism diagnosis were significantly higher if the delivery was a first-time Cesarean section, if the mother had a body mass index of 35 or higher, or if the reason for delivery were a range of fetal problems that made delivery necessary. It was found that boys who were exposed to oxytocin for longer periods of time during labor and received higher total doses of oxytocin had significantly higher odds of developing autism. There were no significant associations of oxytocin dosing and autism noted in female children. As this is the first study to look at any relationship between the dose of oxytocin received during labor and the odds of developing autism, further study needs to be done to determine whether there is any cause and effect relationship. Thus, at this time, there is no recommended change in clinical practice.

The Methylcytosine Dioxygenase Ten-Eleven Translocase-2 (tet2) Enables Elevated GnRH Gene Expression and Maintenance of Male Reproductive Function.

Reproduction depends on the establishment and maintenance of elevated GnRH neurosecretion. The elevation of primate GnRH release is accompanied by epigenetic changes. Specifically, cytosine residues within the GnRH gene promoter are actively demethylated, whereas GnRH mRNA levels and peptide release rise. Whether active DNA demethylation has an impact on GnRH neuron development and consequently reproductive function remains unknown. In this study, we investigated whether ten-eleven translocation (tet) enzymes, which initiate the process of active DNA demethylation, influence neuronal function and reproduction. We found that tet2 expression increases with age in the developing mouse preoptic area-hypothalamus and is substantially higher in a mature (GT1-7) than an immature (GN11) GnRH cell line. GnRH mRNA levels and mean GnRH peptide release elevated after overexpression of tet2 in GN11 cells, whereas CRISPR/cas9-mediated knockdown of tet2 in GT1-7 cells led to a significant decline in GnRH expression. Manipulations of tet2 expression altered tet2 genome binding and histone 3 lysine 4 trimethylation abundance at the GnRH promoter. Mice with selective disruption of tet2 in GnRH neurons (GnRH-specific tet2 knockout mice) exhibited no sign of altered pubertal timing in either sex, although plasma LH levels were significantly lower, and fecundity was altered specifically in adult male GnRH-specific tet2 knockout animals, indicating that tet2 may participate in the maintenance GnRH neuronal function. Exposure to bisphenol A, an environmental contaminant that alters GnRH neuron activity, caused a shift in tet2 subcellular localization and a decrease in histone 3 lysine 4 trimethylation abundance at the GnRH promoter. Finally, evaluation of tet2 protein interactions in GT1-7 cells suggests that the influence of tet2 on neuronal function are not limited to nuclear mechanisms but could depend on mitochondrial function, and RNA metabolism. Together, these studies implicate tet2 in the maintenance of GnRH neuronal function and neuroendocrine control of male reproduction.

Acute Influences of Bisphenol A Exposure on Hypothalamic Release of Gonadotropin-Releasing Hormone and Kisspeptin in Female Rhesus Monkeys.

Bisphenol A (BPA) is an industrial compound with pervasive distribution in the environments of industrialized countries. The U.S. Centers for Disease Control recently found that greater than 90% of Americans carry detectable levels of BPA, raising concern over the direct influences of this compound on human physiology. Epidemiologic evidence links elevated BPA serum concentrations to human reproductive dysfunction, although controlled studies on the acute effect of BPA exposure on reproductive function are limited, particularly in primates. We evaluated the effect of direct BPA exposure on female primate hypothalamic peptide release. Specifically, using a microdialysis method, we examined the effects of BPA (0.1, 1, and 10nM) directly infused to the stalk-median eminence on the release of GnRH and kisspeptin (KP) in mid to late pubertal ovarian intact female rhesus monkeys. We found that the highest level of BPA exposure (10nM) suppressed both GnRH and KP release, whereas BPA at lower concentrations (0.1 and 1nM) had no apparent effects. In addition, we measured BPA in plasma and hypothalamic dialysates after an iv bolus injection of BPA (100 µg/kg). We found a relatively stable distribution of BPA between the blood and brain (plasma:brain ≅ 5:1) persists across a wide range of blood BPA concentrations (1-620 ng/mL). Findings of this study suggest that persistent, high-level exposures to BPA could impair female reproductive function by directly influencing hypothalamic neuroendocrine function.

MeCP2 regulates GFAP expression within the developing brain.

Mutations in MECP2 cause Rett syndrome (RTT), an X-linked neurodevelopmental disorder that primarily affects females. Individuals with RTT have increased glial fibrillary acidic protein (GFAP) expression in the brain. GFAP is an intermediate filament protein that is expressed predominately within astrocytes in the CNS. MeCP2 binds to methylated regions of the GFAP promoter region and suppresses GFAP expression in vitro. Therefore, we wanted to determine if transiently reducing MeCP2 expression would increase GFAP expression in the developing rat brain. Male and female rats received infusions of either MeCP2 or control siRNA targeting the amygdala during the first 3 days of postnatal life. Brains were collected after 6h or 2 weeks following the last infusion. MeCP2 siRNA increased GFAP mRNA and protein within the female, but not the male, amygdala on postnatal day (PN) 2. Two weeks following the infusion, levels returned to normal. MeCP2 siRNA targeting the hypothalamus also increases GFAP mRNA within the female hypothalamus on PN2, suggesting that the regulation is not brain region-specific. It appears that MeCP2 does not regulate all astrocyte markers in the developing female brain, but specifically regulates GFAP expression, as levels of S100ß and vimentin were not altered in the female amygdala at either time point. These data contribute to the idea that the role of MeCP2 differs in the developing male versus female brain. Further elucidating the regulation and function of GFAP can contribute to our understanding of MeCP2 function and perhaps RTT etiology.

Neuroestradiol in the hypothalamus contributes to the regulation of gonadotropin releasing hormone release.

Release of gonadotropin releasing hormone (GnRH) from the medial basal hypothalamus (MBH)/median eminence region (S-ME) is essential for normal reproductive function. GnRH release is profoundly regulated by the negative and positive feedback effects of ovarian estradiol (E2). Here we report that neuroestradiol, released in the S-ME, also directly influences GnRH release in ovariectomized female monkeys, in which the ovarian source of E2 is removed. We found that (1) brief infusion of E2 benzoate (EB) to the S-ME rapidly stimulated release of GnRH and E2 in the S-ME of ovariectomized monkeys, (2) electrical stimulation of the MBH resulted in GnRH release as well as E2 release, and (3) direct infusion of an aromatase inhibitor to the S-ME suppressed spontaneous GnRH release as well as the EB-induced release of GnRH and E2. These findings reveal the importance of neuroestradiol as a neurotransmitter in regulation of GnRH release. How circulating ovarian E2 interacts with hypothalamic neuroestrogens in the control of GnRH release remains to be investigated.

Epigenetic control of gonadotropin releasing hormone neurons.

Epigenetic modifications to the genome, including DNA methylation and histone modifications, occur in response to external stimuli. Reproductive function is highly sensitive to environmental conditions including season, diet, hormonal changes, and exposure to chemical contaminants. GnRH neurons, which play a key role in reproduction, are particularly sensitive to various environmental stimuli. We recently reported that the rhesus monkey GnRH gene exhibits distinct epigenetic changes during embryonic development. More recently, we further found that a similar epigenetic phenomenon occurs across puberty. In this article we highlight recent findings, including those of afferent inputs, to describe the epigenetic control of GnRH circuit development as a link between the environment and reproductive function.

Tonic control of kisspeptin release in prepubertal monkeys: implications to the mechanism of puberty onset.

Previously we have shown that a reduction in γ-amino butyric acid (GABA) inhibition is critical for the mechanism initiating puberty onset because chronic infusion of the GABA(A) receptor antagonist, bicuculline, significantly increased GnRH release and accelerated the timing of menarche and first ovulation in female rhesus monkeys. Because previous studies in our laboratory indicate that in prepubertal female monkeys, kisspeptin release in the medial basal hypothalamus is low, whereas kisspeptin-10 can stimulate GnRH release, we hypothesized that a low level of kisspeptin release prior to puberty onset is due to tonic GABA inhibition. To test this hypothesis we examined the effects of bicuculline infusion on kisspeptin release using a microdialysis method. We found that bicuculline at 1 µM dramatically stimulates kisspeptin release in the medial basal hypothalamus of prepubertal monkeys but had little effect on kisspeptin release in midpubertal monkeys. We further examined whether bicuculline-induced GnRH release is blocked by the presence of the kisspeptin antagonist, peptide 234. We found that inhibition of kisspeptin signaling blocked the bicuculline-induced stimulation of GnRH release, suggesting that kisspeptin neurons may relay inhibitory GABA signals to GnRH neurons. This implies that a reduction in tonic GABA inhibition of GnRH release is, at least in part, mediated through kisspeptin neurons.

Neonatal MeCP2 is important for the organization of sex differences in vasopressin expression.

Several neurodevelopmental disorders are marked by atypical Methyl-CpG-binding protein 2 (MeCP2) expression or function; however, the role of MeCP2 is complex and not entirely clear. Interestingly, there are sex differences in some of these disorders, and it appears that MeCP2 has sex-specific roles during development. Specifically, recent data indicate that a transient reduction in MeCP2 within developing amygdala reduces juvenile social play behavior in males to female-typical levels. These data suggest that MeCP2 within the amygdala is involved in programming lasting sex differences in social behavior. In the present study, we infused MeCP2 or control siRNA into the amygdala of male and female rats during the first three days of postnatal life in order to assess the impact of a transient reduction in MeCP2 on arginine vasopressin (AVP), a neural marker that is expressed differentially between males and females and is linked to a number of social behaviors. The expression of AVP, as well as several other genes, was measured in two-week old and adult animals. Two-week old males expressed more AVP and galanin mRNA in the amygdala than females, and a transient reduction in MeCP2 eliminated this sex difference by reducing the expression of both gene products in males. A transient reduction in MeCP2 also decreased androgen receptor (AR) mRNA in two-week old males. In adulthood, control males had more AVP-immunoreactive (AVP-ir) cells than females in the centromedial amygdala (CMA), bed nucleus of the stria terminalis (BST) and in the fibers that project from these cells to the lateral septum (LS). A transient reduction in MeCP2 eliminated this sex difference. Interestingly, there were no lasting differences in galanin or AR levels in adulthood. Reducing MeCP2 levels during development did not alter estrogen receptorα, neurofilament or Foxg1. We conclude that a transient reduction in MeCP2 expression in the developing male amygdala has a transient impact on galanin and AR expression but a lasting impact on AVP expression, highlighting the importance of MeCP2 in organizing sex differences in the amygdala.

Body weight impact on puberty: effects of high-calorie diet on puberty onset in female rhesus monkeys.

Secular trends toward a declining age at puberty onset with correlated changes in body weight have been reported in economically advanced countries. This has been attributed to excess calorie intake along with reduced physical activity in children. However, because the timing of puberty in humans is also influenced by other factors, such as genetic traits, living conditions, geographical location, and environmental chemicals, it is difficult to distinguish the effect of diet and body size from other factors in a human population. Here we report that feeding juvenile female rhesus monkeys born and raised at the Wisconsin National Primate Research Center with a high-calorie diet results in acceleration of body growth and precocious menarche. The monkeys fed a high-calorie diet also had an elevated body mass index. The most significant treatment effects on circulating hormones were increased leptin and IGF-I levels throughout the experiment. The findings of this study suggest the importance of close monitoring of juvenile feeding behaviors as an important intervention to reduce the prevalence of precocious development and metabolic diseases in adulthood.

Epigenetic changes coincide with in vitro primate GnRH neuronal maturation.

Cellular and molecular mechanisms underlying pulsatile GnRH release are not well understood. In the present study, we examined the developmental changes in intracellular calcium dynamics, peptide release, gene expression, and DNA methylation in cultured GnRH neurons derived from the nasal placode of rhesus monkeys. We found that GnRH neurons were functionally immature, exhibiting little fluctuation in intracellular calcium ([Ca(2+)](i)) and sparse pulses of GnRH peptide release in the first 12 d in vitro (div). By 14-18 div, GnRH neurons exhibited periodic [Ca(2+)](i) oscillations, synchronizing at approximately 60-min intervals and GnRH pulses occurred at approximately 60-min intervals. Interestingly, the total GnRH peptide release further increased after 18 div. Measurement of GnRH mRNA and gene CpG methylation status at 0, 14, and 20 div indicated that mRNA levels significantly (P < 0.05) increased between 14 and 20 div, just as maximal decapeptide release was observed. By bisulfite sequencing across a 5' CpG island of the GnRH gene, we further found that methylation at eight of 14 CpG sites significantly (P < 0.05) decreased between 0 and 20 div. These data indicate that epigenetic differentiation occurs during GnRH neuronal development and suggest that increased GnRH gene expression and decreased CpG methylation status are molecular phenotypes of mature GnRH neurons. To our knowledge, this is the first report that developmental DNA demethylation occurs in postmitotic neurons toward a stable neuronal phenotype.

Sex differences in epigenetic regulation of the estrogen receptor-alpha promoter within the developing preoptic area.

Sex differences in the brain are largely organized by a testicular hormone surge that occurs in males shortly after birth. Although this hormone surge is transient, sex differences in brain and behavior are lasting. Here we describe a sex difference in DNA methylation of the estrogen receptor-alpha (ERalpha) promoter region within the developing rat preoptic area, with males exhibiting more DNA methylation within the ERalpha promoter than females. More importantly, we report that simulating maternal grooming, a form of maternal interaction that is sexually dimorphic with males experiencing more than females during the neonatal period, effectively masculinizes female ERalpha promoter methylation and gene expression. This suggests natural variations in maternal care that are directed differentially at males vs. females can influence sex differences in the brain by creating sexually dimorphic DNA methylation patterns. We also find that the early estradiol exposure may contribute to sex differences in DNA methylation patterns. This suggests that early social interaction and estradiol exposure may converge at the genome to organize lasting sex differences in the brain via epigenetic differentiation.

Mecp2 organizes juvenile social behavior in a sex-specific manner.

Methyl-CpG-binding protein 2 (MeCP2) binds methylated DNA and recruits corepressor proteins to modify chromatin and alter gene transcription. Mutations of the MECP2 gene can cause Rett syndrome, whereas subtle reductions of MeCP2 expression may be associated with male-dominated social and neurodevelopmental disorders. We report that transiently decreased amygdala Mecp2 expression during a sensitive period of brain sexual differentiation disrupts the organization of sex differences in juvenile social play behavior. Interestingly, neonatal treatment with Mecp2 small interfering RNA within the developing amygdala reduced juvenile social play behavior in males but not females. Reduced Mecp2 expression did not change juvenile sociability or anxiety-like behavior, suggesting that this disruption is associated with subtle behavioral modification. This suggests that Mecp2 may have an overlooked role in the organization of sexually dimorphic behaviors and that male juvenile behavior is particularly sensitive to Mecp2 disruption during this period of development.

Sex difference in mecp2 expression during a critical period of rat brain development.

Pervasive developmental disorder is a classification covering five related conditions including the neurodevelopmental disorder Rett syndrome (RTT) and autism. Of these five conditions, only RTT has a known genetic cause with mutations in Methyl-CpG-binding protein 2 (MeCP2), a global repressor of gene expression, responsible for the majority of RTT cases. However, recent evidence indicates that reduced MeCP2 expression or activity is also found in autism and other disorders with overlapping phenotypes. Considering the sex difference in autism diagnosis, with males diagnosed four times more often than females, we questioned if a sex difference existed in the expression of MeCP2, in particular within the amygdala, a region that develops atypically in autism. We found that male rats express significantly less mecp2 mRNA and protein than females within the amygdala, as well as the ventromedial hypothalamus (VMH), but not within the preoptic area (POA) on post-natal day 1 (PN1). At PN10 these differences were gone; however, on this day males had more mecp2 mRNA than females within the POA. The transient sex difference of mecp2 expression during the steroid-sensitive period of brain development suggests that mecp2 may participate in normal sexual differentiation of the rat brain. Considering the strong link between MeCP2 and neurodevelopmental disorders, the lower levels of mecp2 expression in males may also underlie a biological risk for mecp2-related neural disorders.

Discovery and characterization of a cytochrome b5 variant in humans with impaired hydroxylamine reduction capacity.

OBJECTIVES: We have shown that cytochrome b5 (cyt b5), along with its reductase, NADH cytochrome b5 reductase (b5R), is capable of direct xenobiotic biotransformation. We hypothesized that functionally significant genetic variability in cyt b5 could be found in healthy individuals. BASIC METHODS: Cyt b5 cDNAs were prepared from peripheral blood mononuclear cells from 63 individuals. MAIN RESULTS: One individual was heterozygous for a sequence variant in cyt b5 (A178G), with a predicted amino acid substitution of T60A. This variant, when expressed in Escherichia. coli, maintained a similar Vmax for the hydroxylamines of sulfamethoxazole, 4-aminobiphenyl, and 2-amino-l-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), compared with wild type cyt b5, with a modestly increased Km (2 to 3.5-fold) for each substrate. When expressed in a mammalian system (HeLa cells), however, T60A was associated with a 70% reduction in cyt b5 protein expression compared with wild type. mRNA expression for both isoforms were comparable in HeLa cells, and translation of these mRNAs in a rabbit reticulocyte lysate system with inhibited proteasomal machinery were also similar. Incubation of these translated enzymes with uninhibited rabbit reticulocyte lysate, however, indicated greater susceptibility of T60A to proteasomal degradation. CONCLUSIONS: These data indicate that a naturally occurring variant in cyt b5, T60A, leads to modestly altered affinity for hydroxylamine substrates and dramatically reduced cyt b5 expression. Work is underway to determine the prevalence of this and other variants in cyt b5 or b5R in a larger population, and to determine the association of such variants with differences in hydroxylamine reduction in vivo.

Reductive detoxification of arylhydroxylamine carcinogens by human NADH cytochrome b5 reductase and cytochrome b5.

Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of arylhydroxylamine carcinogens, and we further hypothesize that this pathway may be a source of individual variability with respect to cancer susceptibility following 4-ABP or PhIP exposure.

Roles of endogenous ascorbate and glutathione in the cellular reduction and cytotoxicity of sulfamethoxazole-nitroso.

Sulfamethoxazole (SMX) is an effective drug for the management of opportunistic infections, but its use is limited by hypersensitivity reactions, particularly in HIV-infected patients. The oxidative metabolite SMX-nitroso (SMX-NO), is thought to be a proximate mediator of SMX hypersensitivity, and can be reduced in vitro by ascorbate or glutathione. Leukocytes from patients with SMX hypersensitivity show enhanced cytotoxicity from SMX metabolites in vitro; this finding has been attributed to a possible "detoxification defect" in some individuals. The purpose of this study was to determine whether variability in endogenous ascorbate or glutathione could be associated with individual differences in SMX-NO cytotoxicity. Thirty HIV-positive patients and 23 healthy control subjects were studied. Both antioxidants were significantly correlated with the reduction of SMX-NO to its hydroxylamine, SMX-HA, by mononuclear leukocytes, and both were linearly depleted during reduction. Controlled ascorbate supplementation in three healthy subjects increased leukocyte ascorbate with no change in glutathione, and significantly enhanced SMX-NO reduction. Ascorbate supplementation also decreased SMX-NO cytotoxicity compared to pre-supplementation values. Rapid reduction of SMX-NO to SMX-HA was associated with enhanced direct cytotoxicity from SMX-NO. When forward oxidation of SMX-HA back to SMX-NO was driven by the superoxide dismutase mimetic, Tempol, SMX-NO cytotoxicity was increased, without enhancement of adduct formation. This suggests that SMX-NO cytotoxicity may be mediated, at least in part, by redox cycling between SMX-HA and SMX-NO. Overall, these data indicate that endogenous ascorbate and glutathione are important for the intracellular reduction of SMX-NO, a proposed mediator of SMX hypersensitivity, and that redox cycling of SMX-HA to SMX-NO may contribute to the cytotoxicity of these metabolites in vitro.

Adverse effects of EMLA (lidocaine/prilocaine) cream and efficacy for the placement of jugular catheters in hospitalized cats.

EMLA is a lidocaine/prilocaine cream used for topical analgesia in human pediatric patients. The purpose of this study was to establish the safety of EMLA in clinically ill cats, to measure systemic absorption and to determine whether EMLA reduced the need for sedation for the placement of jugular catheters. Thirty-one cats were randomized to either a placebo or EMLA cream group. Cream was applied to a 10 cm(2) area over the jugular vein, with 1h of occlusive dressing. Neither anesthetic was systemically absorbed in any cat, and no adverse clinical signs were observed. Struggling during catheter placement was less in the EMLA-treated cats compared to placebo, but did not reach significance (P = 0.06). Jugular catheters were successfully placed in 60% of EMLA-treated cats and 38% of placebo cats; this difference was not statistically significant and may not justify the added steps of EMLA cream administration for this purpose. However, EMLA does appear to be safe in clinically ill cats, and may be useful for other applications such as for skin mass removal or repeated venepuncture.

Early experience in humans is associated with changes in neuropeptides critical for regulating social behavior.

The formation of social attachments is a critical component of human relationships. Infants begin to bond to their caregivers from the moment of birth, and these social bonds continue to provide regulatory emotional functions throughout adulthood. It is difficult to examine the interactions between social experience and the biological origins of these complex behaviors because children undergo both brain development and accumulate social experience at the same time. We had a rare opportunity to examine children who were reared in extremely aberrant social environments where they were deprived of the kind of care-giving typical for our species. The present experiment in nature provides insight into the role of early experience on the brain systems underlying the development of emotional behavior. These data indicate that the vasopressin and oxytocin neuropeptide systems, which are critical in the establishment of social bonds and the regulation of emotional behaviors, are affected by early social experience. The results of this experiment suggest a potential mechanism whose atypical function may explain the pervasive social and emotional difficulties observed in many children who have experienced aberrant care-giving. The present findings are consistent with the view that there is a critical role for early experience in the development of the brain systems underlying basic aspects of human social behavior.

Unusual dehydroxylation of antimicrobial amidoxime prodrugs by cytochrome b5 and NADH cytochrome b5 reductase.

Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble cytochrome b5 and NAD cytochrome b5 reductase, expressed in Escherichia coli. Kinetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to cytochrome b5 and b5 reductase and by chemical inhibitors of b5 reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain cytochrome b5/b5 reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed cytochrome b5/b5 reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. K(m) values were similar to those for microsomes, and V(max) values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with cytochrome b5 or b5 reductase alone or with cytochrome P450 reductase alone or with cytochrome b5. These results indicate that cytochrome b5 and b5 reductase play a direct role in metabolic activation of DB289 to furamidine.