Tiiu Hansson

Regional Distribution of Cytochrome P‐450 in the Rat Brain: Spectral Quantitation and Contribution of P‐450b,e and P‐450c,d

Abstract: The cytochrome P‐450 (P‐450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P‐450b,e and P‐450c,d to brain P‐450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P‐450b and P‐450c (anti‐P‐450b and anti‐P‐450c, respectively). P‐450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p‐chloroamphetamine‐coupled Sepharose. The yield of P‐450 from whole brain was 90 ± 19 pmol/g of tissue, which is ∼ 1% of the level in liver microsomes from control rats. The amount of P‐450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P‐450 content, with yields of up to 400 pmol/g, whereas the substantia nigra yielded only 16–20 pmol/g. Immunohistochemical studies with anti‐P‐450b and anti‐P‐450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti‐P‐450c. In the cerebellum, both anti‐P‐450b and anti‐P‐450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti‐P‐450b gave weak staining of certain astroglia, but with anti‐P‐450c, there was intense staining of neuronal somata. Western immunoblots with P‐450 isolated from different brain regions confirmed the distribution of P‐450b,e and P‐450c,d observed with immunohistochemistry. Of all the brain regions examined, P‐450b,e was detected only in P‐450 obtained from the cerebellum and P‐450c only in the cerebellum and thalamus. However, quantitation of the P‐450b,e and P‐450c bands on the immunoblots by 125I‐labeled protein A revealed that these forms of P‐450 account for <1% of the P‐450 in the cerebellum and thalamus. This low content of P‐450b and P‐450c was also reflected in a low level of ethoxycoumarin O‐deethylase activity in the cerebellum and thalamus. From these studies, it is concluded that there are multiple forms of P‐450 in the brain and these different forms of P‐450 are highly selectively localized to certain cells. Furthermore, most of the P‐450 in the brain remains uncharacterized.

Species characteristics of the hepatic xenobiotic and steroid biotransformation systems of two teleost fish, Atlantic cod (Gadus morhua) and rainbow trout (Salmo gairdneri)

Groups of Atlantic cod and rainbow trout were treated (ip) with beta-naphthoflavone (BNF), phenobarbital, or peanut oil (controls), and properties of the hepatic xenobiotic and steroid metabolizing enzyme systems were evaluated. In both species, BNF treatment resulted in significant induction of microsomal 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin O-deethylase, biphenyl 4-hydroxylase, and phenanthrene oxidation, especially at the 1,2-position. Immunochemical studies with rabbit IgG prepared against the major BNF-inducible cytochrome P-450 in cod, P-450c, revealed increased amounts of immunoreactive protein in liver slices from both species after BNF treatment. The molecular weight of the induced protein was approximately 58,000 Da, as shown by Western blotting. When titrating biphenyl 4-hydroxylation, however, the antibodies distinguished between the two species, inhibiting the activity of BNF-induced cod 90% and that of rainbow trout 40% at 10 mg IgG/nmol P-450. Furthermore, cytochrome b5 content and UDP-glucuronyltransferase activity were significantly induced only in rainbow trout, whereas the specific content of cytochrome P-450 was significantly increased only in cod. Differences between the two species were observed in the levels of constitutive activities, the amount of induction, and in the regioselectivity of phenanthrene oxidation and androstenedione metabolism. Treatment with phenobarbital showed no effect on any of the parameters investigated in either species. The results show that although there are many common features of the hepatic xenobiotic and steroid biotransformation systems of the two teleosts, certain species characteristics exist in constitutive properties and induction responses.

Influence of biological and environmental factors on hepatic steroid and xenobiotic metabolism in fish: interaction with PCB and β-naphthoflavone

Abstract The degree of induction of hepatic cytochrome P-450 monooxygenases in fish by various chemicals may vary owing to many factors such as sex, sexual maturity, age, season and environmental temperature. In the present investigation the influences of gonadal steroids and water temperature on the inductive response were studied. The data indicate that gonadal steroids and water temperature modulate the response of the cytochrome P-450 system in rainbow trout to PCB and β-naphthoflavone.

Immunohistochemical localization of cytochrome P-450 in the rat brain

Immunohistochemical localization of cytochrome P-450 in the brains of normal untreated male rats has been demonstrated with polyclonal antibodies raised against hepatic P-450C. These antibodies recognize P-450C and D, two closely related forms of P-450. We have identified P-450C,D immunoreactivity (P-450C,D-i) within both neurons and glial cells. Neuronal cell bodies showing P-450C,D-i were present in several forebrain areas, including the olfactory bulb, the caudate nucleus, the cingulate, the frontal and the entorhinal cortex, the hippocampus and the thalamus. In the brainstem, P-450C,D-i neurons were detected in the substantia nigra, the nucleus locus coeruleus, raphe nucleus, reticular formation and the trigeminal nerve nucleus and the dorsal motor nucleus of the vagus. Glial cells containing P-450C,D-i were situated within myelinated fiber tracts in the forebrain (e.g. the corpus callosum, the internal capsule) and the brainstem (e.g. the superior cerebellar peduncle, the medial longitudinal fasciculus). Some, but not all, of the P-450C,D-i glial cells also stained with an antibody against glial fibrillary acidic protein. The present findings represent the first immunohistochemical demonstration of cytochrome P-450 in the brain and suggest that cytochrome P-450C,D is constitutive in neurons and glial cells of most brain regions throughout the rat neuraxis.

Testicular atrophy and loss of nerve growth factor-immunoreactive germ cell line in rats exposed to n-hexane and a protective effect of simultaneous exposure to toluene or xylene

Testicular and germ cell line morphology in rats were studied 2 weeks, 10 months and 14 months after cessation of a 61-day inhalation exposure to 1000 ppm n-hexane. Androgen biosynthetic capacity of testis, testosterone blood concentration, vas deferens morphology and noradrenaline (NA) concentration, epididymal sperm morphology, and fertility were also studied. Severe testicular atrophy involving the seminiferous tubules with loss of the nerve growth factor (NGF) immunoreactive germ cell line was found. Total loss of the germ cell line was found in a fraction of animals up to 14 months post-exposure, indicating permanent testicular damage. No impairment of androgen synthesis or androgen dependent accessory organs was observed. Simultaneous administration of 1000 ppm n-hexane and 1000 ppm toluene, or 1000 ppm n-hexane and 1000 ppm xylene, did not cause germ cell line alterations or testicular atrophy. Toluene and xylene were thus found to protect from n-hexane induced testicular atrophy.

Effects of chronic toluene exposure of central monoamine and peptide receptors and their interactions in the adult male rat

The effects of chronic toluene exposure (CTE) (80 ppm, 6 h/day, 5 days/week, 3 months) were studied on neuropeptide and 5-hydroxytryptamine receptors, on protein phosphorylation levels and on catecholamine levels in various brain regions in the 15-month-old male rat. Behavioral parameters and serum levels of hypophyseal hormones and corticosterone were also analyzed. CTE selectively reduced [3H]neurotensin [( 3H]NT) binding in the basal layers of the orbital cortex. Instead, CTE increased the binding of [3H]etorphine in the nucleus accumbens and of [125I]vasoactive intestinal polypeptide [( 125I]VIP) in the area postrema and hypoglossal nucleus. Acute treatment with the irreversible monoamine receptor antagonist N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) increased the binding of [3H]NT in the orbital cortex in toluene exposed rats as compared with the reduced [3H]NT binding obtained in air exposed rats treated with EEDQ. Furthermore, the EEDQ induced increase in [125I]VIP binding in the area postrema and the hypoglossal nucleus was replaced by a reduced binding of [125I]VIP in EEDQ-treated CTE rats. CTE produced an overall increase in calcium-induced back phosphorylation and an overall decrease in cyclic adenosine monophosphate-induced back phosphorylation in the frontoparietal cortex. Noradrenaline stores tended to be reduced within various hypothalamic subnuclei and the serum prolactin levels were increased following CTE. However, no marked effects of CTE were seen on the behavioral parameters. In conclusion, the regional selectivity of CTE in disturbing [3H]NT and [125I]VIP binding may be due to the demonstrated vulnerability of monoamine-neuropeptide interactions to toluene.

Effects of treated municipal wastewater on the hepatic, xenobiotic, and steroid metabolism in trout

Abstract The effects of treated municipal wastewater on the liver microsomal in vitro metabolism of 4-androstene-3,17-dione, benzo[ a ]pyrene (BP), ethylmorphine (EM), and p -nitroanisole (PNA) in rainbow trout, Salmo gairdneri , were studied. Exposure of fish to waste water for 2 weeks markedly increased the PNA- O -demethylase and BP-hydroxylase activities, while EM- N -demethylase activity was decreased. Wastewater treatment increased the cytochrome P-450 content and NADPH cytochrome c reductase activity when expressed on a per gram liver but not on a per miligram protein basis. LSI and microsomal protein content were significantly higher in wastewater-treated fish when compared to those of controls. In addition, wastewater treatment altered the liver microsomal metabolism of androstenedione. Thus, 6β-hydroxylase activity was decreased in wastewater-treated fish, while 17-hydroxysteroid oxidoreductase activity increased. The results indicate the presence of polycyclic aromatic hydrocarbons in treated municipal wastewater.

Persistent effects of neonatal toluene exposure on regional brain catecholamine levels and turnover in the adult male rat

Effects of neonatal toluene exposure (80 ppm, day 1-7, 6 h/day) have been studied on regional brain catecholamine levels and utilization, and on serum levels of hypophyseal and adrenocortical hormones in the adult male rat. Catecholamine levels were measured by quantitative histofluorimetry in the forebrain and hypothalamus and by high pressure liquid chromatography with electrochemical detection in the substantia nigra. Catecholamine utilization was evaluated from the decrease in catecholamines seen after tyrosine hydroxylase inhibition using alpha-methyl-p-tyrosine methyl ester hydrochloride (alpha MT, 250 mg/kg, i.p., 2 h). Serum levels of thyroid stimulating hormone, corticosterone, aldosterone, prolactin and luteinizing hormone were measured by radioimmunoassays. Neonatal toluene exposure produced a reduction of dopamine levels and utilization selectively in the olfactory tubercle and substantia nigra of the adult rat. Furthermore, neonatal toluene exposure produced a significant reduction in the noradrenaline levels and utilization in the substantia nigra and an increase of noradrenaline utilization selectively in the subependymal layer of the median eminence and of the magnocellular part of the paraventricular hypothalamic nucleus. The serum hormone levels were not significantly influenced by neonatal toluene exposure as evaluated in adulthood. However, the alpha MT induced increase in serum prolactin levels was reduced following neonatal exposure to toluene. Neonatal toluene treatment was also found to alter the responses of the catecholamine neurons to subacute toluene exposure in adulthood. In some of the dopamine nerve terminal systems of the forebrain and in the dopamine cell body containing area of the substantia nigra neonatal toluene exposure appears to have made the dopamine neurons insensitive to adult subacute toluene exposure. In the hypothalamic noradrenaline nerve terminal systems, there were even reversed responses to subacute toluene exposure. The present results indicate that neonatal toluene exposure in doses at the threshold limit value produces persistent changes in dopamine and noradrenaline neurons of the forebrain, hypothalamus and substantia nigra in the presence of a relatively intact neuroendocrine system. In addition, neonatal toluene exposure appears to diminish or even counteract the responses to subacute toluene treatment in adulthood.

Neonatal exposure to toluene: Effects on the development of liver microsomal cytochrome P-450 and serum hormone levels in the rat

Lactating Sprague-Dawley rats and their pups were exposed on postnatal days 1-7, 6 h/day, to 80, 500 and 1000 ppm toluene, respectively, by inhalation. Exposure to 80 ppm toluene decreased the liver microsomal AHH activity and the rate of 7 alpha- and 6 beta-hydroxylation of androstenedione in 8-day-old-pups. On the other hand, neonatal exposure to 500 or 1000 ppm toluene resulted in a significant increase in AHH and 7-ethoxyresorufin O-deethylase activities and in the formation of 16-oxygenated metabolites of androstenedione in 8-day-old animals. Exposure to toluene increased the cytochrome P-450 content at all 3 dose levels in male but not in female pups. Twenty-one days after neonatal exposure no such effects were seen in young animals of either sex. In 56-day-old male rats, however, neonatal exposure to 80 ppm toluene resulted in a decreased rate of 6 beta-hydroxylation of androstenedione and a reduced AHH activity. No such effects were seen in female rats of the same age. Neonatal exposure to toluene affected the body and liver weights in 8-day-old pups of both sexes but had no effect on these parameters in 21-day-old animals of either sex. Exposure to 80 ppm toluene during the neonatal period gave a significantly increased body weight of 56-day-old male but not of female rats of the same age although this treatment increased liver weight in both sexes at this age. Serum testosterone levels were decreased in 21-day-old male rats following neonatal exposure to 80 or 500 ppm toluene and in 56-day-old male rats exposed neonatally to 1000 ppm toluene. In conclusion, exposure to toluene during the first week of life caused significant changes in various liver microsomal cytochrome P-450 dependent enzyme activities in 8-day-old pups, whereas the long-term effects on liver metabolism of the adult animal were small.

Differential expression of multiple forms of cytochrome P-450 in vertebrates: Antibodies to purified rat cytochrome P-450s as molecular probes for the evolution of P-450 gene families I and II

Abstract Polyclonal antibodies against rat cytochrome P-450c (P-450IA1), P-450d (P-450IA2), P-450b (P-450IIB1), P-450h (P-450IIC11) and P-450j (P-450IIE1), were used to probe liver microsomes prepared from species in a number of vertebrate groups ranging from the hagfish, a very primitive vertebrate, to the male rat. Cross-reactivity in Western blots was used to examine the appearance of cytochrome P-450 isozymes structurally related to the rat forms in lower vertebrate groups and to provide information on the evolution of cytochrome P-450 gene families I and II and their regulation in lower vertebrates.