The genetic regulation of liver microsomal CYP2E1 activity among strains of the viviparous fish Poeciliopsis.

CYP2E1 expression was examined within, among, and in F(1) and backcross progeny of strains (P. monacha S68-5; P. viriosa M65-23) of the viviparous fish Poeciliopsis. CYP 2E1 activity varied dramatically in P. monacha, and P. viriosa (3.9+/-0.8 and 9.6+/-1.3 microg/min/mg) as well as the temperature which gave maximal activity (T(0)=25 degrees C and 31 degrees C). F(1) individuals from a crosses between P. monacha and P. viriosa, produced progeny whose CYP2E1 activity segregated into three different groups: (1) phenotypically the same as P. viriosa; (2) intermediate between the two parental strains; and (3) phenotypically the same as P. monacha. When a male P. monacha was crossed with a female P. viriosa 25% of the offspring had an intermediate phenotype and 65% the maternal P. viriosa phenotype. From the same cross, 85% of the females progeny had the maternal phenotype, while 80% of male progeny had the intermediate and paternal phenotype, suggesting an effect of the maternal genome on the F(1) phenotype. Among F(1) fish the T(0) was evenly distributed between parental values. In the backcross of a F(1) female to a male P. viriosa, CZX-6-hydroxylase activity segregated into the same three phenotypes with 60% of the progeny expressing the P. monacha phenotype. From the same cross, 70% of females and 40% of males expressed the P. monacha phenotype. The T(0) in the backcross were evenly distributed between the two parental values and the sex ratio among progeny was different than expected.

Killifish metallothionein messenger RNA expression following temperature perturbation and cadmium exposure.

Metallothionein (MT), a cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals. Induction of MT in mammalian and nonmammalian tissues following heavy metal exposure may serve as a defense mechanism and a biomarker of environmental exposure to chemical stressors such as toxic metals. In this study, MT messenger RNA (mRNA) expression was characterized in male and female nonspawning and spawning killifish (Fundulus heteroclitus) following an 8-day exposure to specific sublethal stressors, which included temperature perturbation (26 degrees C or 10 degrees C) and/or 6 ppb of waterborne cadmium chloride (CdCl2). Hepatic, gill, and intestinal MT mRNA, expressed as copy number per microgram of total RNA, was assessed by reverse transcriptase-polymerase chain reaction and electrochemiluminescence using winter flounder (Pleuronectes americanus) MT complementary DNA primers. Liver, gill, and intestine MT mRNA expression was significantly (P < 0.05) increased in nonspawning killifish exposed to 26 degrees C compared with those exposed to 19 degrees C (control). In addition, a significant (P < 0.05) increase in gill MT mRNA induction was observed in nonspawning killifish exposed to 6 ppb of waterborne CdCl2 compared with controls. The results of this study demonstrate significant MT mRNA induction in nonspawning killifish following short-term exposure to physiological and chemical stressors. Thus, further research may be necessary before the use of killifish MT mRNA induction as a biomarker of environmental chemical stress exposure alone.

Development of a high-throughput process for detection and screening of genetic polymorphisms.

A method is described that uses the ABI PRISM 310 genetic analyzer in conjunction with custom-designed software to identify and classify RAPD products. This methodology will also work well with AFLPs and microsatellite analyses. The methodology uses the ABI PRISM 310's high-throughput (> 500 samples per week) capabilities and in-lane molecular weight standards to efficiently separate and size DNA products. Peak detection, locus classification and export of the data in a form accessible by several genetic analysis programs were accomplished through a custom-written software program (Peaks). Various criteria used by the program to identify and classify loci are described, and their effect on population analyses is examined. Criteria providing an effective, robust determination of population structure are presented.

Multiple pathways of prostate carcinogenesis analyzed by using cultured cells isolated from rats treated with N-methyl-N-nitrosourea and testosterone.

Treatment of rats with N-methyl-N-nitrosourea (MNU) and testosterone results in a high incidence of metastasizing dorsolateral prostate tumors. In previous studies, a high frequency (> or = 70%) of a G35 --> A transition mutation at the second position of codon 12 of the Ki-ras oncogene was found in these tumors. This was confirmed in the study reported here, and the frequency of this mutation appeared similar in tumors induced in four different rat strains, regardless of differences in sensitivity among these strains to the induction of prostate cancers by MNU and testosterone: Wistar Furth (62% incidence of grossly visible prostate tumors) > Lobund Wistar (55%) > Fisher 344 (40%) > Copenhagen (37%). A method was developed to isolate and separately culture epithelial and stromal cells from these rat prostate carcinomas. Of 20 primary cell cultures established from histologically confirmed rat prostate carcinomas, 19 (95%) displayed one or more of the following characteristics: the Ki-ras mutation (17 of 20; 85%), anchorage-independent growth in soft agar at early passage (12 of 20; 60%), or tumorigenicity at later passage (eight of eight; 100%). One epithelial cell culture and all five stromal cell cultures established from prostate tumors had none of these characteristics. Epithelial cultures that had the Ki-ras mutation and grew in soft agar constitute the predominant genotype/phenotype (55%), cultures with the mutation that did not grow in soft agar were less frequent (30%), 10% of the cultures had neither characteristic, and only one grew in soft agar but did not have the mutation. These findings suggest that there are at least two and perhaps more different molecular pathways of prostate carcinogenesis in rats treated with MNU plus testosterone. Furthermore, these data suggest that these pathways and the mechanisms determining strain differences in sensitivity to prostate cancer induction are unrelated.

Rapid estimation of spoilage bacterial load in aerobically stored meat by a quantitative polymerase chain reaction.

We report a quantitative PCR which utilizes primers from a conserved 23S rDNA sequence identified in nine different spoilage bacteria commonly present in meat. The PCR detected the spoilage bacteria by amplifying a specific 207 bp sequence from their chromosomal DNA. Quantification of PCR product by electrochemiluminescence revealed that the concentration of the amplified product was dependent on cycle number and the initial number of bacteria present in the sample. Statistical analysis of the results indicated a correlation coefficient of 0.94 (P < 0.001) between aerobic plate count and QPCR luminosity units.

The effects of different treadmill running programs on the muscle morphology of adult rats.

The biochemical adaptations of different muscle fiber types to endurance training of various intensities and durations have previously been investigated. The objective of this study was to determine the effects of two different endurance training programs on muscle fiber morphology. Twenty-four male Sprague-Dawley rats were randomly assigned to three groups: high intensity/low duration endurance trained (HILD), low intensity/high duration endurance trained (LIHD), or untrained (controls). Following the twelve week training period, muscle fibers of the gastrocnemius muscle of the rats were histochemically classified as type I, type IIa, or type IIb following myosin ATPase staining with pre-incubation at pH 4.6. Muscle fiber type distribution and cross-sectional areas were then determined. Neither HILD nor LIHD rats demonstrated significant differences in fiber type distribution compared to controls. When all fiber types were pooled together and analyzed, there were no differences between the three groups with respect to fiber size. However, when the three fiber types were analyzed individually, HILD animals demonstrated a significant reduction in the size of type IIa fibers while LIHD rats experienced a significant diminution in the size of type I and type IIb fibers. Thus, the morphological adaptations of the muscle fibers in the HILD and LIHD groups were fiber type specific.

Effects of teratogenic exposures to Zn2+, Cd2+, Ni2+, Co2+, and Cu2+ on metallothionein and metallothionein-mRNA contents of Xenopus embryos.

Xenopus laevis embryos were analyzed for metallothionein by silver-saturation assay and metallothionein-mRNA by reverse transcriptase/polymerase chain reaction following exposures to the following metal chlorides at levels that caused > 95% malformations and < 7% mortality: Zn2+ (300 microM); Cd2+ (18 microM); Ni2+ (56 microM); Co2+ (1,800 microM); and Cu2+ (5.6 microM). At the beginning of the exposure (stages 8), metallothionein-mRNA and metallothionein levels averaged 2.0 x 10(6) copies/embryo and 19 pmol/embryo, respectively. In control embryos at stages 26, 36, 42, and 46, metallothionein-mRNA content averaged 9, 37, 104, and 97 copies x 10(6)/embryo, and metallothionein content averaged 6, 11, 15, and 18 pmol/embryo. In Zn(2+) -exposed embryos at the same stages, metallothionein-mRNA content averaged 116*, 11,400*, 3,210*, and 14 copies x 10(6)/embryo and metallothionein content averaged 10, 18*, 46*, and 90* pmol/embryo; in Cd(2+)-exposed embryos, metallothionein-mRNA content averaged 22, 7,170*, 1,783*, and 240 copies x 10(6)/embryo and metallothionein content averaged 8, 14, 33*, and 56* pmol/embryo, respectively (*P < 0.05 versus controls). Exposure-response curves (Cd2+, 1-18 microM; Zn2+, 3-300 microM) indicated that Cd2+ was 3- to 5-times more potent than Zn2+, based on metallothionein-mRNA response at stage 36 and metallothionein response at stage 46. In Ni(2+)-, Co(2+)-, or Cu(2+)-exposed embryos, metallothionein-mRNA and metallothionein contents did not differ significantly from controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of metallothionein mRNA by RT-PCR and chemiluminescence.

A general procedure has been developed for the determination of mRNA expression by reverse transcription polymerase chain reaction (RT-PCR), over a wide concentration range, with quick quantitation of amplified products by luminescence. The discriminating power of this approach is the specific hybridization of PCR product to ruthenium-labeled oligonucleotide probe(s). This method is sensitive enough to detect increases in the formation of PCR product by the 6th cycle. The accumulation of PCR product was successfully modeled with a recursive relationship. This procedure was capable of accurately determining starting template copies over a 9-log dynamic range, with a sensitivity limit of 10(2) copies. Inclusion of an mRNA internal standard (identical to amplified template except for a 6-bp deletion) corrected variabilities in the reverse transcriptase as well as PCR, allowing for the expression of data as mRNA copy number/micrograms total tissue RNA. This procedure was used to detect changes in levels of winter flounder (Pleuronectes americanus) liver metallothionein mRNA. Liver metallothionein mRNA levels ranged from 1.0 x 10(6) copies/micrograms total tissue RNA in control samples to 1.0 x 10(9) copies/micrograms total tissue RNA in samples treated with Cd (a known metallothionein mRNA inducer).

Endurance and resistance exercise induce muscle fiber type specific responses in androgen binding capacity.

This study examined the effects of different exercise training programs on androgen receptor content and receptor affinity to dihydrotestosterone in fast glycolytic (FG) and slow oxidative (SO) skeletal muscle fibers in rats. Twenty-four male Sprague-Dawley rats were equally divided into three groups: control, endurance exercise trained and resistance exercise trained. After the exercise programs were completed, the extensor digitorum longus (EDL), predominantly a FG muscle, and the soleus, predominantly a SO muscle, were isolated, weighted and both androgen receptor content and affinity to dihydrotestosterone were determined. Resistance training evoked a significant (P < 0.05) hypertrophic response in the soleus but not the EDL. Endurance training was not associated with any significant hypertrophy in either the soleus or the EDL. Neither the endurance nor the resistance training program resulted in changes in androgen receptor affinity to dihydrotestosterone. However, alterations in androgen receptor content were noted. The endurance training program resulted in a significant increase in androgen receptor content in the soleus, but no significant difference in the EDL. The resistance training program elicited a significant decrease in androgen receptor content in the soleus, and a significant increase in the EDL. These results indicate that different exercise stimuli induce changes in androgen receptor content that are specific to skeletal muscle fiber type.

The effects of exercise training of different intensities on neuromuscular junction morphology.

Little is known about the effects of exercise training on neuromuscular junction morphology in skeletal muscle. The objectives of this investigation were: 1) to determine if exercise training would elicit changes in neuromuscular junction morphology, 2) to determine if exercise training of different intensities would evoke specific changes in neuromuscular junction morphology, and 3) to determine whether changes in neuromuscular junction structure occur independently of changes in muscle fibre type and size. Twenty-four age and size matched male Sprague-Dawley rats were randomly assigned to three groups: high-intensity trained (HIT), low-intensity trained (LIT), or untrained. Neuromuscular junction morphology of the soleus muscle was determined via immunofluorescent staining. Presynaptic acetylcholine vesicles were visualized with SV-2 antibody in conjunction with fluorescein isothiocyanate labelled secondary secondary antibody. Postsynaptic acetylcholine receptors were identified with rhodamine labelled alpha-bungarotoxin. Laser scanning microscopy was used to produce images of synapses, which were used to quantitate the following: total area of SV-2 and alpha-bungarotoxin staining, density of acetylcholine vesicles and receptors, structural complexity, and synaptic coupling. To visualize nerve terminal branching, a smaller number of neuromuscular junctions were stained with C-2 antibody, which reacts with a neurofilament epitope, in conjunction with fluorescein isothiocyanate labelled secondary antibody. Total length of branching, number of branches, average length of branches, and ratio of secondary to primary branches per neuromuscular junction were determined. Citrate synthase activity, fibre type composition and fibre cross-sectional areas of the soleus muscle were assessed to determine the presence of a training effect in that muscle. Results indicate that training did induce hypertrophy of the neuromuscular junction that was independent of muscle hypertrophy. Although the HIT and LIT groups exhibited similar hypertrophic responses of the neuromuscular junction, the HIT group displayed more dispersed synapses than the LIT group. Neither exercise training program, however, resulted in altered densities of acetylcholine vesicles or receptors, nor did training significantly change synaptic coupling. Nerve terminal branching was also affected by exercise training. Neuromuscular junctions from the HIT group demonstrated a greater total length of branching, average length per branch, and number of finer, or secondary, branches than those of the LIT group.

Isolation and characterization of a cDNA for osteopontin-k: a kidney cell adhesion molecule with high homology to osteopontins.

Screening of a bovine renal cDNA library with MAbs resulted in the isolation of a 1447 bp cDNA. This cDNA (pBk2.1) was sequenced and shown to contain an open reading frame with a putative protein of 261 amino acids, with a molecular weight of 29,573 (minute leader sequence) and a hydrophobic leader sequence of 16 amino acids. pBk2.1 was shown to share a high level of nucleic acid sequence homology over portions of its sequence to human, porcine, mouse, and rat osteopontins (40-60%). The peptide (osteopontin-k) had a potential glycosylation site (Asn-X-Ser/Thr), a GRGDS receptor binding region, a high level of asparagine residues, and a high abundance of acid amino acids characteristic of osteopontin-like cell adhesion molecules. The N-terminal amino acid region of pBk2.1 (the first 82 amino acids) and 42 amino acids at the C terminus had the highest level of homology with the osteopontins at 86%. The middle portion of the peptide had greatly reduced homology, ranging from 50% (amino acids 83-174) to 12% (amino acids 175-219). There were also deletions and additions of sequence in osteopontin-k that were not found in the other osteopontins. The homologies suggest that these proteins are highly related and may be derived from a common gene by alternative splicing. A 678 bp cRNA probe constructed from pBk2.1, containing a region with low homology to the osteopontins (amino acids 183-219 with less than 20% homology, plus amino acids 220-261 and untranslated sequence), was used in northern blots and RNAse protection assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrosodiethylamine metabolism in the viviparous fish Poeciliopsis: evidence for the existence of liver P450pj activity and expression.

Several species of fish from the genus Poeciliopsis differ dramatically in their response to the carcinogen N-nitrosodiethylamine (NDEA). The differential induction of tumors among genotypes exposed to NDEA may, in part, result from differences in liver cytochrome P450pj activity (the piscine equivalent of mammalian P450j). Evidence for the existence of cytochrome P450pj activity and mRNA expression has been found in several Poeciliopsis genotypes (species and strains). Biochemical evidence suggests that a microsomal cytochrome P450 enzyme catalyzes the metabolism of NDEA to acetaldehyde and other intermediates in Poeciliopsis. This reaction was inhibited by carbon monoxide, and required molecular oxygen and reducing equivalents (NADPH). Differences were found in maximal activity as well as temperature optima among genotypes. Poeciliopsis, a livebearing fish from desert streams of northwestern Mexico, appears to have thermal optima for cytochrome P450pj activity between 25 and 30 degrees C depending on the genotype. Western blot analysis (using anti-rat P450IIE1 antibodies) detected a 55-60 kd band in microsomes isolated from rat and Poeciliopsis. Using a 49mer probe specific for rat cytochrome P450j, Northern blots revealed a 3.3 kb mRNA from livers of a Poeciliopsis genotype and rat, but none in muscle mRNA from either organism. S1 nuclease protection assays, using the same probe, revealed that a mRNA fragment protected by the probe against digestion was induced on exposure of the whole organism to ethanol (via uptake from the aquatic environment). The assays also demonstrated that ethanol treatments both induced and suppressed this mRNA, depending on concentration and exposure time.

Characterization of monoclonal antibodies specific to bovine renal vitamin D hydroxylases.

Monoclonal antibodies (MAbs) have been produced which recognize specific epitopes on bovine renal mitochondrial vitamin D3 1 alpha- and 24-hydroxylases. Renal mitochondria cytochrome P-450s were partially purified to 0.5-2 nmol/mg by Emulgen 911 and cholate solubilization, followed by chromatography on a 2-(4,6-dichloro-O-biphenyloxy)ethylamine HBR affinity column. Reduced carbon monoxide difference spectra determined that this preparation contained 0.5-2 nmol P-450/mg protein. This preparation contained both 1 alpha- and 24-hydroxylase activities, and Eadie-Hofstee plots of product formation as a function of substrate concentrations have maximum velocities of 1.4 and 4 pmol product/30 min.mg protein and Km values of 690 and 1300 nM, respectively. Bovine renal hydroxylases were isolated by immunoprecipitation from this partially purified P-450 preparation with a polyclonal antibody specific for rat liver microsomal cytochrome P-450 RLM5. This polyclonal antibody immunoprecipitated both 1 alpha- and 24-hydroxylase activities as well as renal mitochondrial cytochrome P-450, as determined by reduced CO spectra. Bovine renal mitochondrial components were immunoisolated and used to immunize BALB/c mouse spleen cells in vitro. MAbs then produced were screened for 1) immunoisolation of renal mitochondrial hydroxylase activity from a partially purified preparation, 2) immunohistochemical detection of antigen in renal proximal tubule cells, and 3) immunoquantitation of renal hydroxylases in a solid phase sandwich (enzyme-linked immunosorbent assay) and by 4) Western blot analysis. MAbs were isolated with specifically immunoprecipitated 1 alpha-hydroxylase activity, 24-hydroxylase activity, or both. In 10 micron sections of bovine kidney, antibodies detected antigen only in proximal tubule cells on the basal surface, which is rich in mitochondria. No antigen was detected in sections of pancreas or liver. In the solid phase sandwich enzyme-linked immunosorbent assay, MAbs detected 1 alpha and 24-hydroxylases only in renal mitochondria and not in liver microsomes or adrenal gland mitochondria. In a Western blot, MAbs specific for epitopes expressed on both hydroxylases detected a single band(s) at 52,000-53,000 daltons. Apparently it is not possible to discriminate between hydroxylases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots. By these criteria, MAbs have been generated which are specific to epitopes expressed on bovine renal mitochondrial 1 alpha- and 24-hydroxylases.

Oxidative stress limits vitamin D metabolism by bovine proximal tubule cells in vitro.

When bovine proximal tubule cells are placed in primary culture, they are subject to elevated oxidative stress which acts to limit the expression of mitochondrial vitamin D3 1 alpha- and 24-hydroxylase activities. This increased oxidative stress was demonstrated by increased production of cell and mitochondrial membrane lipid hyperperoxides (LOOH). This increased production was prevented by the addition of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Cell and mitochondrial membrane LOOH increased from 1 to 2 pmol/mg protein on the day of plating to 70-90 pmol/mg protein after 6 days in culture. Pretreatment of cultures with BHA and BHT resulted in membrane LOOH of 15-20 pmol/mg protein after 6 days. Mitochondrial LOOH production was greater than total cell LOOH after 6 days. The increase in cellular oxidative stress was paralleled by decreases in both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. Mitochondrial hydroxylase activities were inversely proportional to the increase in mitochondrial membrane LOOH production. Mitochondrial cytochrome P-450 content, determined spectrophotometrically, was decreased over time in culture. Mitochondrial cytochrome P-450 content determined by a specific polyclonal antibody in an enzyme-linked immunosorbant assay also decreased over time in culture. Specificity of polyclonal antibodies, raised against rat liver microsomal cytochrome P-450 RLM5, was demonstrated by the immunosequestration of both 1 alpha- and 24-hydroxylase activities from a partially purified preparation of renal mitochondrial cytochrome P-450. BHA showed the loss of 1 alpha- and 24-hydroxylase activities and mitochondrial P-450 content measured by all criteria. These experiments indicate that oxidative stress-mediated changes in hydroxylase activities are mediated directly by changes in hydroxylase content and not at distal sites. A partially purified preparation of bovine proximal tubule mitochondrial cytochrome P-450, with purified renal ferredoxin, ferredoxin reductase, and NADPH, expressed both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. LOOH, derived from mitochondrial membranes of 5-day-old cultures, when added to this mixture, caused a dose-dependent decrease in both activities. These experiments suggested that an increase in mitochondrial LOOH production resulted in a loss of 1 alpha- and 24-hydroxylase activities. 1 alpha-Hydroxylase was more sensitive to the effects of LOOH treatment than 24-hydroxylase. At a ratio of LOOH:P-450 of 5:1 (molar), all 1 alpha-hydroxylase activity was lost but 50% of the 24-hydroxylase activity remained.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of bovine renal mitochondrial cytochrome P-450 with antioxidants.

The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguiaretic acid (NDGA), benzyl sulfoxide (BS), ferulic acid (FA), caffeic acid (CA), dimethyl sulfoxide (Me2SO), protocatechuic acid (PCA), and P-450 inhibitor metyrapone all acted to slow the previously noted loss of vitamin D3 1 alpha-and 24-hydroxylase activities in cultured bovine proximal tubule cells. The slowing of the loss of hydroxylase activities by antioxidants was increased by culturing cells in 5% O2 vs 19% O2. These same antioxidants also directly inhibited 1 alpha- and 24-hydroxylase activities. For a single antioxidant, or metyrapone, Ki's for inhibition of both hydroxylases were equal, ED50's for stabilization of both hydroxylase activities were equal, and Ki's and ED50's were not significantly different. These antioxidants prevented tert-butylhydroperoxide (tert-BOOH)-mediated proximal tubule cell death at concentrations, i.e., 0.1 mM, which were effective in stabilizing hydroxylase activities. When added together, the antioxidants H2SeO3, uric acid, and trolox c gave slight stabilization of hydroxylase activities without inhibiting hydroxylase activities. Singly, these antioxidants did not stabilize or directly inhibit hydroxylase activities. This antioxidant combination augmented BHA- or BHT-mediated stabilization of both hydroxylase activities independent of any effects on inhibition. But the most potent antioxidants which acted to stabilize hydroxylase activities in culture also directly acted to inhibit hydroxylase activities. Antioxidant effects were additive for both inhibition and stabilization of hydroxylase activities. Stabilization of hydroxylase activities was dissociated from inhibition in the presence of maximal FA, CA, and BHA or FA, CA, and BHT combinations. Bovine renal mitochondrial cytochrome P-450 levels decreased in cultured bovine proximal tubule cells to nondetectable levels by 8 days in culture. When cultures were treated with BHA and BS, mitochondrial P-450 levels were almost twofold greater than in untreated controls. Percentage changes in mitochondrial P-450 levels closely paralleled percentage changes in hydroxylase activities elicited by antioxidant treatment regimes. Antioxidants which were effective inhibitors of hydroxylase activities in cultured bovine proximal tubule cells were also effective in inhibiting hydroxylase activities in isolated proximal tubule mitochondria, supplemented with a NADPH-generating source. Ki's for inhibition of hydroxylase activities were very similar in cultured cells and in isolated mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)

Bovine renal mitochondrial vitamin D3 hydroxylases: regulation of in vitro activities by inhibitors and antioxidants.

The regulation of bovine renal 1 alpha- and 24-hydroxylase activities was examined in primary bovine proximal tubule cell cultures. Maximal 1 alpha- and 24-hydroxylase activities in primary bovine proximal tubule cultures ranged from 1.5-1.8 and 2.0-2.7 pmol/min X 10(6) cells, respectively. The apparent Km was 795 nM for 1 alpha-hydroxylase activity and 1130 nM for 24-hydroxylase activity. 1 alpha- and 24-hydroxylase activities decreased in primary culture after cell plating. Activities decreased both as a function of cell number and as a function of the culture dish. 1 alpha-Hydroxylase activity decayed with a t1/2 of 37 h, while 24-hydroxylase activity decayed with a t1/2 of 45 h. Decreasing cell densities, at which cells were plated, increased the t1/2 for the decay of both activities [t1/2 = 21 h at 5,000 cells/cm vs. t1/2 = 37 h at 25,000 cells/cm for 1 alpha-hydroxylase (P greater than 0.001); t1/2 = 33 h at 5,000 cells/cm vs. t1/2 = 45 h at 25,000 cells/cm for 24-hydroxylase, (P greater than 0.0001)]. Direct addition of 0.25 mM metyrapone inhibited 1 alpha-hydroxylase activity by 33% and 24-hydroxylase activity by 51%. Long term incubation of cell cultures with 0.25 mM metyrapone resulted in a slowing in the loss of both hydroxylase activities, but did not stop the decay. 1 alpha-Hydroxylase activity in 4-day metyrapone-treated cultures was 35% higher than in 4-day untreated cultures. 24-Hydroxylase activity was increased 42% in treated cultures vs. that in untreated cell cultures. Both 1 alpha- and 24-hydroxylase activities were inhibited by direct addition of antioxidants. 1 alpha-Hydroxylase activity was directly inhibited 74% by the addition of 0.1 mM butylated hydroxyanisole (BHA), 69% by the addition of 0.1 mM butylated hydroxytoluene (BHT), and 56% by the addition of 0.05 mM benzyl sulfoxide (BS). 24-Hydroxylase activity was also directly inhibited 72% by 0.1 mM BHA, 55% by 0.1 mM BHT, and 73% 0.05 mM BS. There was no significant difference between the inhibition of either hydroxylase by each antioxidant. Antioxidant mixtures increased the inhibition of hydroxylase activities above that with single antioxidant. The addition of 0.1 mM BHA and 0.05 mM BS to cultures resulted in 100% inhibition of 24-hydroxylase activity and 95% inhibition of 1 alpha-hydroxylase activity. The results were very similar when 0.01 mM BS and 0.1 mM BHT were added to cultures, i.e. 100% and 91% inhibition of 24- and 1 alpha-hydroxylase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of cultured bovine adrenocortical zona glomerulosa cell 17-hydroxylase activity by ACTH.

The induction of steroid 17-hydroxylase activity was examined in primary cultures of bovine adrenocortical zona glomerular cells. 17-Hydroxylase activity was determined by assaying the metabolism of [3H]pregnenolone to [3H]17-hydroxypregnenolone by high performance liquid chromatography (HPLC). Conversion of [3H]pregnenolone to [3H] progesterone and [3H]17-hydroxypregnenolone to [3H]17-hydroxyprogesterone was prevented by the addition of cyanoketone, an inhibitor of 3 beta-hydroxysteroid dehydrogenase. ACTH increased adrenal zona glomerulosa 17-hydroxylase activity 55-fold to 1750 pmoles/10(4) cells/2 h, an activity equivalent to that of ACTH-treated zona fasciculata cells. Maximal induction was seen after 4 days exposure to ACTH. The dose of ACTH which gave half-maximal induction was 0.25 nM. Previously, ACTH had been demonstrated to suppress bovine glomerulosa conversion of deoxycorticosterone to aldosterone by a steroid-induced oxygen-derived free radical process that was prevented by antioxidants. This process resulted in loss of the terminal cytochrome P-450 enzyme involved in aldosterone biosynthesis. However, induction of 17-hydroxylase activity is not affected by antioxidants or the addition of steroids, e.g. cortisol. ACTH-mediated induction of bovine zone glomerulosa 17-hydroxylase activity and suppression of glomerulosa aldosterone production results in effective conversion of functional glomerulosa cells to functional fasciculata cells. The actions of ACTH on glomerulosa 17-hydroxylase activity support the hypothesis of a steroid-induced functional zonation of the adrenal cortex.