Effect of chromate ion on the membrane of established human cells as measured by uptake of a permeant lipophilic cation.

Cells from the established human cell line NHIK3025 concentrate the permeant lipophilic cation triphenylmethylphosphonium (TPMP+) against a concentration gradient, indicating the existence of an electrical potential across the cell membrane (interior negative). Cells exposed to potassium chromate or dichromate (7.7 mumol Cr/l) for 2 h subsequently showed reduced uptake of TPMP+, whereas similar exposure to a trivalent chromium salt (7.7 mumol/l chromic chloride) had similar uptake of TPMP+ as control cells. These preliminary results suggest that a primary effect of chromate on these cells is to reduce the electrical potential across the cell membrane.

Corticosteroids and lung surfactant levels in adult male rats.

Following lung instillation in adult male rats of 3.4 mumol hexavalent chromium (K2Cr2O7) dissolved in 0.5 ml of 0.9% NaCl, increased levels of lung surfactant could be detected after 48 h. The blood serum concentration of corticosterone was elevated in these animals. Blood serum thyroxine and triiodothyronine showed an initial increase after lung instillation of hexavalent chromium followed by a decline. Metabolism of testosterone by the alveolar macrophages to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol was reduced 6 and 12 h after the K2Cr2O7 instillation, which was also associated with damage of lung cell function and decreased uptake by the alveolar macrophages of Candida albicans particles. As early as 12 h after s.c. administration of 400 micrograms dexamethasone/100 g body wt, increased levels of lung surfactant could be measured. At this time the lungs showed no signs of cellular damage, and metabolism of testosterone as well as uptake of Candida albicans particles by the alveolar macrophages were normal. Lower s.c. doses of dexamethasone did not result in raising the levels of lung surfactant in 12 h. Within 12 h after s.c. administration of large doses of testosterone, dihydrotestosterone or dehydroepiandrosterone no measurable effects on the levels of lung surfactant could be measured. Since animals treated with dexamethasone (200 micrograms/100 g body wt) or long-acting synthetic ACTH (100 micrograms i.m. Synacthen Depot/100 g body wt) for 5 days after lung instillation of K2Cr2O7 had extremely high levels of lung surfactant, it is concluded that the corticosteroids in adult rats may help to create augmented surfactant levels following lung intoxication. This could proceed via stimulation of surfactant production and reduction of surfactant removal. Different aspects of lung surfactant metabolism are discussed.

Steroid hormone biosynthesis by a sesterterpene pathway in the rat and rabbit testis.

Rat and rabbit testis preparations were incubated with [4-14C]cholesterol and 23,24-dinor-[7 alpha-3H]5-cholen-3 beta-ol, the latter being a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin-layer chromatography and crystallised to constant specific activity. It was found that rat and rabbit testis can utilise 23,24-dinor-5-cholen-3 beta-ol to produce testosterone. The tritium/carbon-14 ratios in the testosterone and androstenedione isolated indicated that these tissues differentiated between the two substrates. This finding is supported by the observation that, on stimulation with HCG, the tritium/carbon-14 ratios in the testosterone isolated were increased compared to the controls. The results of further experiments implied that, while the biosynthesis of testosterone from cholesterol occurred in the rat testis mitochondrial fraction, its biosynthesis from 23,24-dinor-5-cholen-3 beta-ol occurred in the microsomal fraction.

Metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol in alveolar macrophages from rat lung--II. Effects of intratracheal instillation of 3.4 mumol K2Cr2O7.

Activity of the steroid 5 alpha-reductase in pulmonary alveolar macrophages from adult male rats has been investigated in vitro. Intratracheal instillation of 3.4 mumol K2Cr2O7 lowered the enzyme activity within 6 h, and the reduction was significant on the subsequent 2, 4 and 7 days. The activity of this enzyme was significantly decreased only 6 and 24 h after instillation when measured in the 800 g supernatant fraction of whole lung. Instillation of 3.4 mumol K2Cr2O7 increased serum levels of corticosterone. Serum levels of triiodothyronine and thyroxine decreased except for a transient increase 3 h after the K2Cr2O7 instillation. Subcutaneous administration of 200 micrograms dexamethasone/100 g b.wt, 200 micrograms/100 g b.wt of testosterone, 17 beta-hydroxy-5 alpha-androstane-3-one (5 alpha-DHT), dehydroepiandrosterone or corticosterone had no effect on the 5 alpha-reductase activity of the pulmonary alveolar macrophages within 12 h. The combined treatment with dexamethasone s.c. and intratracheal instillation of 3.4 mumol K2Cr2O7 reduced the steroid 5 alpha-reductase activity in the pulmonary alveolar macrophages to about 25% of controls. Measurement of the steroid 5 alpha-reductase activity in pulmonary alveolar macrophages as an index of lung damage when exposed to toxic material is discussed.

Biological reactivity of different crystalline forms of titanium dioxide in vitro and in vivo.

The biological reactivity of two crystalline forms of titanium dioxide, rutile and anatase, has been compared in in vitro and in vivo assays. Rutile and anatase induced similar effects, and both had a very low biological activity in comparison to alpha-quartz. Rutile samples containing trace amounts of nickel or chromium had an activity similar to that of pure rutile.

Circulating steroids in male rats following inhalation of n-alcohols.

The effect of inhaled methanol, ethanol, n-propanol and n-butanol on male reproductive function as measured by the serum concentration of circulating T (testosterone) and LH (luteinizing hormone) has been investigated. The animals were exposed to concentrations of these alcohols equal to the current threshold limit values in industry (methanol: 200 ppm, ethanol: 1000 ppm, n-propanol, n-butanol: 50 ppm) 6 h a day for up to 1 week. A significant depression in the concentration of circulating T was found after the first 6 h exposure to the respective alcohols with restoration after a recovery period of 18 h, except in rats exposed to n-butanol where a depression of 48% could still measured. The concentration of LH was within the normal range in all experimental groups whereas corticosterone was increased after exposure to n-butanol. Exposure for 1 week was not associated with any significant inability of the rat testis to produce T.

Concentration-dependent effects of potassium dichromate on the cell cycle.

Hexavalent chromium is found to be a strong mutagen, and it also is a potential carcinogen in man. DNA flow cytometry, growth measurements, and determinations of mitotic index show that 1-2 microM K2Cr2O7 produces a prolongation of the G2 phase of the cell cycle in NHIK 3025 cells. By increasing the chromate concentrations (greater than 2 microM K2Cr2O7) the cells are also arrested in G2 phase. We have found, using synchronized cells and measuring cell cycle time, that the most chromate-sensitive part of the cell cycle is S phase. This phase is also somewhat prolonged, and the cells became arrested in early S phase at high toxic K2Cr2O7 concentrations (8 microM). Our results thus indicate that K2Cr2O7 has an effect within S phase--maybe on DNA/RNA synthesis--and also interferes with processes necessary for progression through the G2 phase.

Metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol in alveolar macrophages from rat lung.

5 alpha-Reduction of testosterone was observed in lung cells obtained by bronchoalveolar lavage (greater than 95% macrophages) from the rats. This activity was inhibited by progesterone and corticosterone. Production of 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol from testosterone was higher in rat pulmonary alveolar macrophages than by the 800 g supernatant fraction of whole lung homogenate from the same animals. Alveolar macrophages from rats treated with the 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5 alpha-androstane-3-one (5 mg/100 g b.w., s.c.) showed decreased metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol 4 h after treatment. This metabolism was also decreased in alveolar macrophages from rats exposed to potassium dichromate by intratracheal instillation. When bovine alveolar macrophages were incubated with potassium dichromate, 5 alpha-reduction of testosterone decreased significantly. The function of steroid 5 alpha-reduction in alveolar macrophages is currently not known.

Effects of fixing and staining on images of tropomyosin Mg-paracrystals.

Tropomyosin Mg-paracrystals have been studied by positive and negative staining with uranyl acetate and sodium phosphotungstate, either unfixed or fixed with glutaraldehyde. Fixation causes changes in some of the paracrystal bands. Some of the bands are more intense when a positively-charged staining ion is used than when a negatively-charged ion is used, in both negative and positive-staining techniques. This result rules out the suggestion that negative staining is not affected by the charge of the stain in tropomyosin, and agrees with findings in other specimens. Therefore it is unlikely that any simple parameter based on residue size can be used to predict the image intensity from the amino acid sequence for tropomyosin. These staining patterns cast doubt on the earlier interpretation of a prominent white band in the paracrystal as an overlap of alpha-helical molecular ends.

Circulating concentrations of testosterone, luteinizing hormone and follicle stimulating hormone in male rats after inhalation of methanol.

Male mature rats were examined for alterations in circulating free testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) after inhalation of methanol vapour in a dynamic system for up to 6 weeks at doses ranging 200 ppm to 10,000 ppm. The most extensive effects were observed after exposure to 200 ppm of methanol for 6 weeks with serum testosterone concentrations being 32% of the controls. A significant change in LH concentration after exposure to 10,000 ppm of methanol for 6 weeks was also demonstrated. FSH remained unchanged throughout these experiments. Such exposure did not change the elimination rate of testosterone from blood which indicated effects on the testicular synthesis of testosterone.

Ileal metabolism in vitro of testosterone to 4-androstene-3 alpha, 17 beta-diol.

Testosterone metabolism to 4-androstene-3 alpha, 17 beta-diol by the 800 g supernatant fraction of ileum from male and female rats was investigated. Ileal production of this testosterone metabolite was higher in mature female animals than in mature males. This difference could be eliminated by administration of large doses estradiol-17 beta to mature male rats. Immature animals showed low ileal production of 4-androstene-3 alpha, 17 beta-diol before being weaned from their mother.

Response of bovine alveolar macrophages in vitro to welding fume particles.

Alveolar macrophages (AM) from bovine lungs were exposed in culture to manual metal are (MMA) welding fume particles, chromium (Cr), UICC chrysotile A or anatase for 17-20 hr. All the welding particle samples were more cytotoxic to AM than to anatase. Particles from the welding of mild steel with a rutile-coated electrode were less cytotoxic than those produced with a basic-coated electrode. Particles from the welding of stainless steel were slightly more cytotoxic, and much of this activity was probably due to CrVI. Selective release of N-acetyl-beta-glucosaminidase (beta-NAG) was only detected after exposure of AM to chrysotile. Supplementation of the incubation medium with 10% serum increased the viability of all exposed AM cultures, an effect not produced by serum albumin alone. Incubation of particle samples with dipalmitoyl phosphatidylcholine (DPPC) prior to addition to AM reduced the cytotoxicity of the "rutile" welding particles and of chrysotile.

Metabolism in vitro of testosterone (T) to 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) and 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha) by the 800 g supernatant fraction of ileum from rats.

The 800 g supernatant fraction of ileum from rats was incubated with testosterone and production of 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha,17 beta-diol measured. No difference in this production could be determined between ileum from mature female and mature male animals. Ileum from immature rats staying with their mothers from day 12 to day 21 showed increased 5 alpha-reduction of testosterone on days 15, 17 and 21. Ileal metabolism to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha,17 beta-diol was increased in immature animals on day 25 of extrauterine life when consuming rat chow and water ad libitum from day 21. This increase could not be demonstrated on day 28. Immature and mature animals treated either with long-acting ACTH or dexamethasone showed increased ileal conversion of testosterone to the two 5 alpha-reduced metabolites determined. Ileum from mature male rats injected, subcutaneously, with arabinosylcytosine every 8th h for 2 days exhibited increased metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha,17 beta-diol from the 1st to the 6th day after the last injection of arabinosylcytosine. Different aspects of intestinal metabolism of testosterone are discussed.

Biochemical and cytological studies of rat lung after inhalation of methanol vapour.

Rats were examined for changes at the lung surface and in lung tissue after inhalation of methanol vapour for up to 6 weeks at doses of up to 10 000 ppm. No significant changes were found in any of the parameters measured. Methanol vapour therefore does not appear to exert appreciable toxicity at these exposure levels towards rat lung.

Structure requirements for glucocorticoid growth of a human cell line (NHIK 3025).

The human cell line NHIK 3025 has a cytoplasmic dexamethasone receptor. When these cells are exposed to glucocorticoids, the cell cycle time is prolonged. The structural requirements for this effect were investigated by measuring cell number after 4 days exposure to different glucocorticoid analogues at concentrations of 10(-6) M and 10(-7) M. Growth inhibition at 10(-7) M required the 4-5 double bond, the 3,20 ketone--and the 11 beta, 17 alpha and 21 hydroxygroups of the glucocorticoid structure, e.g. cortisol, dexamethasone and prednisolone. Other synthetic glucocorticoids like bimetrazol and triamcinolone acetonide were also active at this dose whereas corticosterone, lacking the 17 alpha hydroxygroup, was only active at 10(-6) M. The effect was steroid specific and could be inhibited by anti-cortisol antiserum or by glucocorticoid antagonists.

In vitro metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol by the rat intestine.

The metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol by the 800 g supernatant fraction by different parts of the gastrointestinal tract from male rats was investigated. This metabolism tended to be higher in immature than in mature animals. Administration of dexamethasone or long-acting ACTH to immature and mature rats increased testosterone metabolism to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol by ileum tissue. No such effect could be observed following administration of progesterone, estradiol, prolactin, LH or FSH in mature animals. Development of the gastrointestinal tract from the immature to the mature stage was associated with augmented metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol in the ileum.

Hexavalent chromium and ascorbic acid interaction on proliferation of the human cell line NHIK3025.

When cells from the human cell line NHIK3025 were cultured for 4 days, with hexavalent chromium (K2Cr2O7) at a concentration of 8 . 10(-7)M, the relative cell number was reduced to 38%. This effect of hexavalent chromium was abolished if ascorbic acid and chromate were added simultaneously to the incubation medium, but not if ascorbic acid was added 24 h prior to chromate addition. Dehydroascorbic acid was not able to reduce the effect of K2Cr2O7 either when added simultaneously or when added 24 h prior to chromate exposure. Therefore, the cells could only be protected against the toxic effect of hexavalent chromium when ascorbic acid was added at the same time as K2Cr2O7.

Effects in vitro of medroxyprogesterone acetate on steroid metabolizing enzymes in the rat: selective inhibition of 3 alpha-hydroxysteroid oxidoreductase activity.

The effects of 6 alpha-methyl-17 alpha-acetoxy-4-pregnene 3,20-dione (MPA) on the activity of different steroid metabolizing enzymes in vitro were investigated in several organs in the rat. MPA seems to be a potent inhibitor of 3 alpha-reduction of 17 beta-hydroxy-5 alpha-androstan-3-one (Dht) in homogenates of the testis, ovary, epididymis, prostate, kidney and the adrenal glands. In testicular homogenates MPA acts like a competitive inhibitor of the 3 alpha-reduction of Dht, with Ki of 0.42 [microM]. MPA seems to be a selective inhibitor of 3 alpha-hydroxysteroid oxidoreductase in numerous organs. Steroid metabolizing enzymes like 5 alpha-reductase, 7 alpha-hydroxylase, 3 beta-hydroxysteroid oxidoreductase and 17 beta-hydroxysteroid oxidoreductase were not inhibited by MPA under the conditions of incubation employed in these studies.

Stimulation in vivo of testicular 7 alpha-hydroxylase activity in immature rats by LH and hCG.

Testicular C19-steroid 7 alpha-hydroxylase activity rises during puberty in the rat. Hormonal mechanisms responsible for this rise are unknown. The present study is to the best of our knowledge the first to report successful stimulation of testicular 7 alpha-hydroxylase in immature rats. Administration of crude and purified hCG every day for 3 or more days to immature rats in doses of 3 I.U. lead to significant stimulation of testicular 7 alpha-hydroxylase activity. Purified preparations of subunits of hCG did not stimulate 7 alpha-hydroxylase activity. Stimulation of testicular 7 alpha-hydroxylase was, however, recorded when purified subunits of hCG were allowed to recombine and then administered to the animals. Several preparations of LH failed to cause stimulation of 7 alpha-hydroxylase when administered to immature rats. Treatment of immature rats with mixtures of LH and FSH, LH and prolactin or LH together with FSH and prolactin was also ineffective in this respect. Large doses of highly purified ovine-LH, rat LH and human LH were, however, able to stimulate testicular 7 alpha-hydroxylase activity in immature rats. Preparations comprising hybrids of ovine-LH and hCG (oLH alpha + hCG beta, hCG alpha + oLH beta) stimulated testicular 7 alpha-hydroxylase activity in immature rats. Administration of the same amounts of a hybrid preparation of human and ovine-LH (oLH alpha + hLH beta) to immature rats was ineffective in this respect. Administration of testosterone or estradiol to immature rats lead to suppression of testicular 7 alpha-hydroxylase activity. Combined treatment of such rats with hCG and testosterone or with estradiol and hCG augmented testicular 7 alpha-hydroxylase activity to the same degree as that of animals treated with hCG only.

C-19-steroid 7 alpha-hydroxylation by rat testes. Isolation and identification of: 7 alpha, 17 beta-dihydroxy-5 alpha-androstan-3-one, 5 alpha-androstan-3 alpha, 7 alpha, 17 beta-triol and 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol.

From incubations of testosterone with rat testicular homogenates in the presence of a NADPH-generating system, the following 7 alpha-hydroxylated metabolites could be isolated and identified: 7 alpha, 17 beta-dihydroxy-4-androsten-3-one (7 alpha-hydroxy-testosterone), 7 alpha-17 beta-dihydroxy-5 alpha-androstan-3-one (7 alpha-hydroxy-Dht), 5 alpha-androstan-3 alpha, 7 alpha, 17 beta-triol (7 alpha-hydroxy-3 alpha-A'DIOL) and 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol (7 alpha-hydroxy-3 beta-A'DIOL). To our knowledge this is the first demonstration of the formation of 5 alpha-reduced-7 alpha-hydroxylated metabolites of testosterone in the male gonad. These 5 alpha-reduced-7 alpha-hydroxylated metabolites could also be isolated after incubations of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-A'DIOL) with testicular homogenates in the presence of A NADPH-generating system. Measured as the sum of 7 alpha-hydroxy-testosterone, 7 alpha-hydroxy-Dht. 7 alpha-hydroxy-3 alpha-A'DIOL and 7 alpha-hydroxy-3 beta-A'DIOL formed using testosterone as substrate, total 7 alpha-hydroxylase activity was six times higher in testes of mature rats than in testes from animals 23 days old. With 3 alpha-A'DIOL as substrate total 7 alpha-hydroxylase in the mature testis was about three times greater than in the sexually immature testis.