Effect of single neonatal or repeated benzpyrene exposure on the serotonin content of immune cells in young male rats

In earlier experiments single benzpyrene treatment of newborn rats caused strong alterations in the endorphin content of adult rats' immune cells. In the present experiments young (4-6 weeks old) male rats were studied for demonstrating the effect of the single neonatal or repeated (neonatally and at weanling) benzpyrene exposure on the serotonin content of immune cells (blood lymphocytes, monocytes, granulocytes; peritoneal fluid lymphocytes, mast cells, monocytes and granulocytes, thymic lymphocytes). Flow cytometric analysis showed that 50 microg benzpyrene treatment of five-week-old animals was ineffective after 5 days and this was the situation four weeks after single neonatal (20 microg) benzpyrene exposure. However, the repeated treatment of neonatally benzpyrene exposed 4 weeks old animals after 5 days resulted in elevated blood and thymic lymphocyte serotonin amount and in one index (peritoneal monocyte-granulocyte group) reduced serotonin content. This means that neonatal benzpyrene treatment does not influence directly the serotonin content (production or transport) of immune cells (unlike to the endorphin content) however, sensitizes them to a following benzpyrene exposure. The results widen the list of harmful effects (influencing steroid receptor binding, sexual behavior and immune cells' endorphin content) of perinatal benzpyrene exposure.     

Effect of neonatal benzpyrene imprinting on the brain serotonin content and nocistatin level in adult male rats

Single neonatal treatment (imprinting) with 20 microg benzpyrene results in significant increase of the brain serotonin level in the striatum, while in the other four regions (cortex, brainstem, hippocampus, hypothalamus) when measured in adults can be detected. The nocistatin level of cerebrospinal fluid (CSF) significantly decreases, while there is no change in the plasma nocistatin level. The results call attention to the comprehensive imprinting effect of benzpyrene, which in addition to receptorial, hormonal and sexual behavioral disturbances causes lasting differences in the brain serotonin and nocistatin levels, probably influencing mood and pain tolerance.     

Endorphin content of white blood cells and peritoneal cells in neonatally benzpyrene treated adult rats

White blood cells of rats (lymphocytes, monocytes, macrophages, granulocytes and mast cells) contain beta-endorphin. Two months after a single neonatal benzpyrene treatment (imprinting) there is an elevated level of immunoreactive endorphin in the blood and peritoneal cells of female animals and blood cells of males. The endorphin content decreased in the peritoneal cells of males. In the blood, the granulocytes of female, and the lymphocytes of male rats contained the highest amount of endorphin. In the peritoneal fluid also the granulocytes of females contained the highest amount of endorphin, in contrast to males, where the endorphin content of cells decreased and the lowest level of it was present in the lymphocytes. The experiments justify that benzpyrene treatment can durably influence endorphin levels of white blood cells and gives new data to the already known lifelong health destroying effects of perinatal benzpyrene exposition (alterations of hormone receptor binding capacity and sexual behavior).     

The purification of human DNA fragments containing benzpyrene adducts

DNA was isolated from human diploid lung epithelial cells treated in culture with [3H]benzpyrene. The DNA contained one covalently bound benzpyrene group per 38 kb and it was digested with a series of restriction endonucleases giving decreasing fragment sizes, and also with DNAase I to 96% acid solubility. The digests were applied to octyl-Sepharose columns under conditions which promote hydrophobic interaction of the benzpyrene groups on the DNA with the octyl groups in the column matrix. Separation of fragments without and with benzpyrene groups was achieved with successive high salt and ethanediol washes. As DNA fragment size is decreased, more DNA-associated benzpyrene is eluted with ethanediol. Under these conditions, DNA from untreated cells is totally removed in the high salt wash and free benzpyrene metabolites are retained on the column. The separation of DNA fragments with covalently-bound hydrophobic benzpyrene groups, from less modified or unmodified DNA will facilitate examination of the distribution of benzpyrene adducts in defined regions of the human genome.     

Neuromediator provision of the organs of immune system in benzpyrene intoxication]

The density of adrenergic innervation and relative content of catecholamines were investigated in rat lymphoid organs under different type of benzpyrene treatment. It was found that both ante- and postnatal influence of toxicant leads to a decrease of neurotransmitter providing of the thymus, spleen, mesenterial, iliac and popliteal lymph nodes. On the contrary, when mixed ante- and postnatal benzpyrene influence has place, the adrenergic innervation density and relative content of catecholamines are increased. We suppose that benzpyrene treatment of pregnant animals has specific "training" effect for monoaminergic systems of foetus and causes the increase of neurotransmitter providing of immunocompetent organs in conditions of repeat body and toxicant meeting.     

A reliable,sensitive, and convenient radioactive assay for benzpyrene monooxygenase

A new radioactive assay for benzpyrene monooxygenase has been developed, characterized, and compared to the fluorescent assay generally employed. The radioactive assay is based on a single extraction which effectively separates metabolites from remaining substrate. This assay is linear within reasonable ranges of time and protein concentration and responds as expected to inhibitors and an inducer of benzpyrene monooxygenase activity. The activity measured with the present assay is about twice that measured with the fluorescent assay. Furthermore, the radioactive assay can be scaled down so that it is at least as sensitive as the fluorescent assay.     

The environmental pollutant aromatic hydrocarbon, benzpyrene has deleterious effect on hormone receptor development

In this review, works to clear the effect of perinatal and pubertal benzpyrene imprinting to the steroid hormone receptors of adult animals are summarized. There is a comparison between the perinatal and adolescent effects and between the effects on the receptors of males and females. On the basis of the experiments benzpyrene is a general and dangerous imprinter which can influence durably the development of different steroid receptors given directly to the animals studied (perinatally) of given to the nursing mothers or influencing the offspring generations of the perinatally treated parents or grandparents. The results call attention to the outstanding deleterious effects of benzpyrene pollution.     

Binding capacity of rat liver glucocorticoid receptor in different periods after single neonatal benzpyrene treatment (imprinting)

Newborn rats of both sexes were treated (imprinted) with 20 microg of benzpyrene. Two hours, 2 days, 1, 2, 3 weeks, 1 month and 2 months after imprinting the liver glucocorticoid receptors were studied for binding of dexamethasone. Two-hour and 2-day values were not appreciable. One week after treatment the receptor's affinity was extremely low both in control and treated treated animals. Two weeks after imprinting a significant difference in density (lower) and affinity (higher) was observed between the male treated and control animals. At 3 weeks and one month the binding capacity of treated and control animals was equal however, at 2 months Bmax of males increased and that of females decreased significantly in the neonatally benzpyrene treated animals. This means that for the development of perinatal imprinting effect a long time is needed, and the effect is manifested after a period of lability.     

Reappraisal of the liver benzpyrene hydroxylase synthesized de novo after treatment of rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene

The dynamics of the inductive effects of MC and TCDD upon rat liver microsomal benzpyrene hydroxylase and the main properties of the de novo synthesized hemoproteins have been compared. The inadequacy of expression of the enzyme activity per total cytochrome P-448 content has been established. It was concluded that TCDD microsomes have a relatively low content of benzpyrene hydroxylase with a higher molecular activity than the enzyme from MC microsomes.     

Effect of single neonatal treatment with the soy bean phytosteroid,genistein on the sexual behavior of adult rats

Hormonal imprinting develops during the perinatal critical period, when the target hormone meets the yet unmatured receptor. As a consequence of imprinting the receptor accomplishes its maturation reaching the binding capacity characteristic to adults. In this period in the presence of foreign molecules similar to the target hormone faulty imprinting may occur with life-long consequences. Soy bean contains phytosteroids which can mimic estrogen effects. In the present experiments single genistein (20 microg) or combined genistein + benzpyrene (20 microg) treatments were done neonatally and the sexual behavior of male and female adult animals was studied. Genistein significantly increased the lordosis quotient of females, which was compensated by neonatal benzpyrene treatment. Genistein also enhanced the sexual activity of males, and this was significantly not reduced by parallel benzpyrene treatment. The results show that neonatal genistein exposure can imprint sexual activity for life and the presence of a second imprinter can modify genistein's behavioral effect.     

Michaelis--Menten kinetic analysis of the hepatic microsomal benzpyrene hydroxylase from control, phenobarbital- and methyl-3-cholanthrene-treated rats.

The sensitive fluorimetric assay for hydroxy-3-benzpyrene (3-OH-BP) described by Dehnen et al., was used to study the effect of microsomal membrane concentration of the benzpyrene hydroxylase activity. Microsomes from phenobarbital (PB) and methyl-3-cholanthrene (3-MC)-treated rats were used in comparison with the microsomal fraction from control animals. At very low protein concentration, benzpyrene hydroxylase follows as Michaelis--Menten type kinetics. When the concentration of microsomal membrane is higher than a minimal value (+/- 6 mug protein/ml) the Km increases with increasing concentration of protein due to competitive inhibition by reversible and non-specific binding of the substrate. The Ki's for such a binding have been calculated. Pretreatment of rats with 3-MC selectively shortens the time linearity, decreases the Ks value, and has no effect on Vmax, while the administration of PB prolongs the time linearity, decreases Vmax and does not modify the Ks. 3-MC and PB specifically act on cytochrome P-450 and do not modify the physico-chemical properties of the microsomal membrane as measured by the non-specific binding of benzpyrene (BP).     

Prolonged impact of five imprinters on the serotonin content of white blood cells and mast cells of weanling rats: outstanding effect of benzpyrene and chlorpheniramine

In previous experiments, treatment at weaning or adult age with endorphin, serotonin or an antihistamine (late hormonal imprinting) durably influenced the serotonin content of white blood cells and mast cells of rat. In the present experiments, five molecule (approved imprinters in other indexes) were studied for imprinting effect of immune cells, 3 weeks after a single treatment at weaning. Three steroid hormone-like molecule (vitamin D3, mifepristone and dexamethasone) were ineffective (except dexamethasone in 1/4 indexes), while benzpyrene (aromatic hydrocarbon) and chlorpheniramine (H1-receptor blocker antihistamine) were highly effective (5/6 and 4/4 respectively). The results indicate: (1) a prolonged (late imprinting) effect of a single treatment with certain molecules acting at receptor level; (2) non-generality of late imprinting, and (3) the very extensive effects of benzpyrene, which in earlier experiments was one of the strongest imprinter at receptorial and behavioral level at any periods of life studied.     

Direct and transgenerational effect of benzpyrene treatment at adolescent age on the uterine estrogen receptor and thymic glucocorticoid receptor of the adult rat

Hormonal imprinting develops perinatally at the first encounter between the maturing receptor and the target hormone, helping the normal accomplishment of receptor maturation. In the presence of hormone excess or foreign molecules able to bind to the maturing receptor, faulty imprinting takes place, which disturbs the normal receptor function for life. Earlier experiments demonstrated that the effect of faulty perinatal benzpyrene imprinting of the steroid hormone receptors is transmitted to the progeny generations. In certain organs which are maturing later (such as the uterus) imprinting can be executed at adolescence. In the present experiments pubertal benzpyrene imprinting caused a durable decrease in female's estrogen receptor density. The transgenerational effect of this type of imprinting was also studied. The pubertal imprinting of the parents was transgenerationally transmitted to the offspring generation in which--without further treatment--the density (Bmax) of the uterine estrogen receptors was significantly higher than that in the controls. There were measurable effects neither in the affinity (Kd) of uterine estrogen receptors nor in the Kd and Bmax of the male thymus glucocorticoid receptors. The experiments call attention to the profound and comprehensive imprinting effect of the environmental pollutant benzpyrene.     

Comparative studies on the interaction of benzpyrene with cells derived from poikilothermic and homeothermic vertebrates. II. Effect of temperature on benzpyrene metabolism and cell multiplication

The effect of incubation temperature on cell multiplication and on the efficiency of benzpyrene (BP) metabolism to water-soluble derivatives was compared in cell cultures derived from three poikilothermic and three homeo-thermic vertebrate species. The fish cells grew optimally at about 20°C and the amphibian and reptilian cells at about 30°C, and in general, these cells multiplied over broader ranges of temperature than the mouse, hamster or chick cells. In each cell system, the maximum temperature supporting efficient BP metabolism exceeded the maximum temperature supporting cell growth by 4 to 8°, but the range of temperatures supporting near-maximal BP metabolism was also considerably broader in the poikilothermic than in the homeothermic vertebrate cultures.     

Cellular responses to methylcholanthrene; the role of benzpyrene hydroxylase in the binding of polycyclic hydrocarbon to cytosol macromolecules in fetal rat liver explants

Fetal rat liver was maintained in culture over a period of several days using a simple expiant technique. The addition of 3-methylcholanthrene to the expiants caused a reproducible “induction” of benzpyrene (BP) hydroxylase. In preincubated explants, a 4- to 6-fold elevation in enzyme activity was obtained within 24–36 hr.     

Effect of inducers and inhibitors of monooxygenase on the hydroxylation of prostaglandins in the guinea pig. Evidence for several monooxygenases catalyzing omega- and omega-1-hydroxylation.

The incubation of prostaglandins (PG's) with liver microsomes from guinea pigs treated with inducers of monooxygenase (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzo[alpha]pyrene (benzpyrene), or a mixture of chlorinated biphenyls (Aroclor 1254)) exhibited marked elevation of 19-hydroxylation of PGE1, PGE2, PGA1, and PGA2 without affecting significantly 20-hydroxylation. However, with respect to effects on hydroxylation of a variety of xenobiotics, benzpyrene and Aroclor treatments differed markedly; whereas Aroclor treatment elevated the demethylation of ethylmorphine, benzphetamine, and p-chloro-N-methylaniline (PCMA), benzpyrene treatment had no effect on demethylation of ethylmorphine and only a marginal effect on that of PCMA. Both inducers elevated benzpyrene hydroxylation. By contrast, treatment with phenobarbital did not affect the hepatic microsomal PG's hydroxylation, although the hydroxylation of benzpyrene and the demethylation of ethylmorphine, benzphetamine, and PCMA were enhanced. Also, the hydroxylation of PG's by kidney cortex microsomes was not affected by either benzpyrene or Aroclor treatment. Inhibitors of monooxygenase were used to help delineate the type of monooxygenases induced. At low levels of alpha-naphthoflavone (ANF), benzpyrene hydroxylation in control- and Aroclor-treated guinea pigs was only little affected; by contrast, the same concentration of ANF markedly inhibited benzpyrene hydroxylation in benzpyrene-treated guinea pigs. On the other hand, metyrapone was most inhibitory in control guinea pigs. Support for the conclusion that benzpyrene induces in the guinea pig a hepatic monooxygenase with different characteristics than that found in control animals was provided by the observation that ANF (10 MICROM) inhibited PGE1 hydroxylation more pronouncedly in liver microsomes from benzpyrene-treated than from Aroclor-treated guinea pigs or controls. In addition, in benzpyrene and Aroclor-treated guinea pigs, ANF inhibited the (omega-1)-hydroxylation more pronouncedly than that of omega-hydroxylation. By contrast, metyrapone appeared to inhibit omega-hydroxylation more effectively than (omega-1)-hydroxylation. These results indicate that in the guinea pig, hydroxylation of PG's at the omega (20-) and omega-1 (19-) positions is catalyzed by different monooxygenases and that the inducers tested affect several hepatic monooxygenases with different specificities toward xenobiotics; however, with respect to PG's only the enzyme(s) involved in the 19-hydroxylation is affected.     

Kinetic studies of benzpyrene and hydroxybenzpyrene metabolism.

The kinetics of benzypyrene (BP) metabolism were examined in liver microsomes, and in accordance with the results of Hansen and Fouts [9] exhibited curcilinear Lineweaver-Burk plots. The problem was exacerbated in microsomes of 3-methylcholanthrene (MC-ms) treated rats. The Km for BP, measuring hydroxybenzypyrene (OHBP) appearance was about 0.3 muM in MC-treated adult rats and about 1.0 muM in untreated rats. These values were obtained using a substrate range of 0.2-2.0 muM benzpyrene, 20 mug of microsomal protein/ml and a 3 min assay time. With longer assay times and with higher microsomal protein concentrations curvilinear reciprocal plots were obtained. This was found to be due to a combination of three factors, namely non-specific binding of BP to the microsomes, rapid depletion of substrate, and further metabolism of hydroxy products. At 100 mug microsomal protein/ml about 50% of added BP was non-specifically bound to the microsomes in the range of 0.2-2.0 muM BP. Addition of albumin to the medium (1 mg/ml) greatly enhanced the BP hydroxylase activity but only slightly increased the amount of BP remaining in the medium after sedimentation of the microsomes by centrifugation. 3-OHBP, one of the phenolic products of BP metabolism was found to be metabolized to a non-fluorescent products(s); the Km for this compound was similar to that for BP. Differences were seen in the Vmax rates of BP disappearance and OHBP appearance. Disappearance of BP is several fold faster than OHBP appearance and has a larger Km. The latter may be due to the need to use higher amounts of protein and to allow depletion of enough substrate to make measurements significantly reproducible or the higher Km may reflect a composite value for different routes of BP metabolism.     

Interactions of heme with hepatic microsomal mono-oxygenase. Effect on benzpyrene hydroxylation.

The addition of heme (1-10 muM) to liver microsomes from phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated male rats increased the rate of benzpyrene (BP) hydroxylation by about 20-40%. On the other hand, protoporphyrin IX caused only inhibition of BP hydroxylation. There was no increase of enzymatic activity by heme when solubilized preparations of liver microsomes were used. This suggested the possibility that an apo-cytochrome P-450 was present in intact microsomes. Higher concentrations of heme inhibited BP hydroxylation by either intact or solubilized microsomes. The inhibition by heme with solubilized microsomal preparations was noncompetitive, "mixed-type". However, with intact microsomes, the lack of linearity, precluded the determination of the type of inhibition. To examine possible effects of heme on the binding of BP to microsomal cytochrome P-450, the spectrum elicited by the addition of BP to microsomes was obtained in the presence or absence of added heme. The addition of heme to liver microsomes produced a marked increase in the trough (419-420 nm) of the difference spectrum formed by the subsequent addition of BP. These findings would suggest that heme increased the binding of BP to microsomes. However, the possibility that BP merely displaces the bound heme of the microsomes to yield, as expected, a trough at 413-416 nm (the addition of heme to microsomes yields a peak of 413-416 nm, unpublished) cannot be ruled out. Nevertheless, independent of our understanding of the mechanism involved in the spectral interactions between heme and BP with liver microsomes it is clear that an effect at their binding site(s) must have been elicited by the presence of both compounds.     

Benzpyrene hydroxylase activity in hepatic microsomal and solubilized systems containing rabbit or rat cytochrome P-448 or P-450

Treatment of adult, male rabbits and rats with 3-methylcholanthrene results in the formation of hepatic microsomal cytochrome P-448. In the rat, this occurs coincidently with an increase in hepatic microsomal benzpyrene hydroxylase activity. In the rabbit, benzpyrene hydroxylase activity is decreased following treatment with 3-methylcholanthrene. Benzpyrene hydroxylase activity in solubilized, reconstituted mixed-function oxidase systems containing rat cytochrome P-448 is about seven times higher than in systems containing rabbit cytochrome P-448. Evidence obtained by spectral analysis suggests that rabbit P-448 is combined with a type I compound. Residual ¹⁴C-3-methylcholanthrene does not appear to be responsible for the differences observed between rat and rabbit cytochrome P-448.     

Benzpyrene groups bind preferentially to the DNA of active chromatin in human lung cells.

The cells of the bronchial epithelium of man are targets for benzo(a)pyrene carcinogenesis. When cultures of these cells, and of non-target fibroblasts, are exposed to [3H]-benzo(a)pyrene, we find that the epithelial cells metabolise and bind to DNA far greater amounts of benzpyrene than do fibroblasts. By analysis of nuclei of benzpyrene-treated cells for sensitivity to limited digestion with pancreatic DNase I, we have shown that benzpyrene groups bind initially to the DNA of expressed (DNase I sensitive) regions of chromatin in both cell types. Covalent binding of benzpyrene groups to non-expressed (DNase I resistant) regions follows rapidly in the target epithelial cells. These maintain high levels of carcinogen adducts in their DNA. In fibroblasts, benzpyrene group binding to non-expressed DNA occurs more slowly and active removal of adducts from the DNA is evident.