In vitro and in vivo activity of 1-O-hexadecylpropanediol-3-phospho-ganciclovir and 1-O-hexadecylpropanediol-3-phospho-penciclovir in cytomegalovirus and herpes simplex virus infections.

Human cytomegalovirus (HCMV) and herpes simplex virus (HSV) can cause a wide variety of clinical manifestations in man. Ganciclovir (GCV) is effective against HCMV infection when administered by the intravenous route and may be used orally in large doses for prophylaxis of HCMV infections in organ transplantation patients and in AIDS patients. In previous studies with acyclovir (ACV), we found that covalent attachment of an alkyl glycerol phosphate moiety greatly increased oral bioavailability and increased antiviral activity against hepatitis B virus. Adducts of ACV with alkyl propanediol phosphate were more active than the alkyl glycerol phosphate analogue in vitro in 2.2.15 cells, which constitutively produce hepatitis B virus. To see if this strategy would work for two other poorly absorbed nucleoside analogues, we synthesized 1-O-hexadecylpropanediol-3-phospho-GCV (HDP-P-GCV) and 1-O-hexadecyl-propanediol-3-phospho-penciclovir (HDP-P-PCV), and evaluated the in vitro antiviral activity, selectivity and oral antiviral activity of both compounds versus GCV or PCV in mice infected with HSV-1 or HDP-P-GCV versus murine cytomegalovirus (MCMV). HDP-P-GCV is orally active in both MCMV and HSV-1 infection in mice with antiviral activity equivalent to (HSV-1) or greater than oral GCV (MCMV). Oral HDP-P-PCV was more active than PCV orally versus intranasal HSV-1 infection in mice.

Alkylglycerol prodrugs of phosphonoformate are potent in vitro inhibitors of nucleoside-resistant human immunodeficiency virus type 1 and select for resistance mutations that suppress zidovudine resistance.

Phosphonoformate (foscarnet; PFA) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), but its use for the treatment of HIV-1 infection is limited by toxicity and the lack of an orally bioavailable formulation. Alkylglycerol-conjugated prodrugs of PFA (1-O-octadecyl-sn-glycero-3-PFA [B-PFA]) having sn-2 substituents of hydrogen (deoxybatyl-PFA [DB-PFA]), methyl (MB-PFA), or ethyl (EB-PFA) are more-potent inhibitors of wild-type HIV-1 in vitro than unmodified PFA and are orally bioavailable in mice. We have evaluated the activities of these compounds against a panel of nucleoside-resistant HIV-1 variants and have characterized the resistant variants that emerge following in vitro selection with the prodrugs. Except for an HIV-1 variant encoding the K65R mutation in RT that exhibited 3.3- to 8.2-fold resistance, the nucleoside-resistant viruses included in the panel were sensitive to the PFA prodrugs (<3-fold increase in 50% inhibitory concentration), including multinucleoside-resistant variants encoding the Q151M complex of mutations or the T69S[SA] insert. Viruses resistant to the PFA prodrugs (>10-fold) were selected in vitro after 15 or more serial passages of HIV-1 in MT-2 cells in escalating prodrug concentrations. Mutations detected in the resistant viruses were S117T, F160Y, and L214F (DB-PFA); M164I and L214F (MB-PFA); and W88G and L214F (EB-PFA). The S117T, F160Y, and M164I mutations have not been previously identified. Generation of recombinant viruses encoding the single and double mutations confirmed their roles in prodrug resistance, including 214F, which generally increased the level of resistance. When introduced into a zidovudine (AZT)-resistant background (67N 70R 215F 219Q), the W88G, S117T, F160Y, and M164I mutations reversed AZT resistance. This suppression of AZT resistance is consistent with the effects of other foscarnet resistance mutations that reduce ATP-dependent removal of AZT monophosphate from terminated template primers. The favorable activity and resistance profiles of these PFA prodrugs warrant their further evaluation as clinical candidates.

In vitro anti-HIV-1 activity of sn-2-substituted 1-O-octadecyl-sn-glycero-3-phosphonoformate analogues and synergy with zidovudine.

Monoalkyl ether lipid analogues of foscarnet (phosphonoformate, PFA) exhibit substantially greater in vitro antiviral activity than unmodified PFA against human immunodeficiency virus type 1 (HIV-1). Our previous studies indicate that the length of the alkyl chain must be 14-22 carbons for optimal antiviral activity. To further evaluate the structure-activity relationship, we prepared 1-O-octadecyl-sn-glycerol analogues of PFA with various substitutions at the sn-2 position of glycerol and determined the effect of structure on in vitro antiviral activity and selectivity against HIV-1 in MT-2 and CD4-expressing HeLa cells (HT4-6C). We also studied combinations of zidovudine with PFA, 1-O-octadecyl-2-O-methyl-sn-glycero-3-PFA, or 1-O-octadecyl-sn-glycero-3-PFA and calculated their combination index values against HIV-1 in HT4-6C cells. Alkyl substitutions of one to four carbons at the sn-2 position of glycerol showed optimal antiviral activity. Both alkyl ether lipid analogues were strongly synergistic with zidovudine over a wide range of drug ratios and concentrations. 1-O-octadecyl-sn-glycerol analogues of PFA have selective antiviral properties and warrant further evaluation as potential antiretroviral drugs.

Synthesis and antiviral evaluation of 1-O-hexadecylpropanediol-3-P-acyclovir: efficacy against HSV-1 infection in mice.

We synthesized, 1-O-hexadecylpropanediol-3-P-acyclovir, an orally bioavailable lipid prodrug of acyclovir and evaluated it for in vitro and in vivo activity against herpes simplex virus infections. Although 1-O-hexadecylpropanediol-3-P- acyclovir was less active in vitro than acyclovir, on a molar basis it was 2.4 times more active orally in preventing mortality from acute HSV-1 infection in mice. In vitro, 1-O-hexadecylpropanediol-3-P-acyclovir was also more active than acyclovir in a thymidine kinase negative mutant strain of HSV-1 (DM21) and had somewhat higher activity in cytomegalovirus infection in vitro due to it's ability to bypass thymidine kinase.

Alkylthioglycerol prodrugs of foscarnet: synthesis, oral bioavailability and structure-activity studies in human cytomegalovirus-, herpes simplex virus type 1- and human immunodeficiency virus type 1-infected cells.

In a previous study, we reported that 1-O-octadecyl-sn-glycero-3-foscarnet (ODG-PFA) was 40 to 93 times more potent than free foscarnet (PFA) in human cytomegalovirus (HCMV)-, herpes simplex virus type 1 (HSV-1)- and human immunodeficiency virus type 1 (HIV-1)-infected cells. To evaluate the effect of substituting a 1-S-alkyl thioether for a 1-O-alkyl ether, we synthesized a series of PFA conjugates of 1-S-alkyl-sn-thioglycerols with varied 1-S-alkyl chain lengths. To establish structure-activity relationships we measured the in vitro antiviral activity of liposomal formulations of the drugs in cells infected with HCMV, HSV-1 or HIV-1. The optimum 1-S-alkyl chain length in the series was 16 to 18 carbon atoms. We compared the antiviral activity of 16- and 18-carbon alkyl thioglycerol versus alkylglycerol prodrugs and did not observe any significant differences in their antiviral activities. The series' most active member, 1-S-octadecyl-sn-glycero-3-foscarnet (ODSG-PFA) was 56-, eight- and 45-fold more active than PFA in HCMV-, HSV-1- and HIV-1-infected cells in vitro. The oral absorption of PFA and 1-S-octadecyl-sn-thioglycero-3-PFA was compared in mice by measuring plasma levels of 14C after oral administration of radiolabelled compounds. The peak plasma level of 14C was sevenfold higher following administration of [14C]ODSG-PFA than following an equimolar dose of [14C]PFA. Area-under-the-curve was 23-fold greater for ODSG-PFA than for PFA. Like previously reported alkyloxyether-lipid PFA conjugates, alkylthioether conjugates provided enhanced antiviral activity and oral bioavailability. However, S-ether conjugates may be metabolized differently than O-ether conjugates. More detailed in vivo pharmacokinetic evaluation of the alkyl-thioether-PFA conjugates is required.

Alkoxy propane prodrugs of foscarnet: effect of alkyl chain length on in vitro antiviral activity in cells infected with HIV-1, HSV-1 and HCMV.

The identification of more effective and less toxic foscarnet (PFA) analogs for antiviral therapy would be useful. We recently synthesized 1-O-octadecyl-sn-glycero-3-phosphonoformic acid (ODG-PFA) and noted a 93-fold increase in its anti-HCMV activity relative to PFA. In addition, the antiviral activity of ODG-PFA in herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type-1 (HIV-1) infected cells was increased 40-fold relative to PFA (Hostetler et al., 1996. Antiviral Res. 31, 59). To evaluate structure-activity relationships further, we synthesized alkoxypropyl esters of foscarnet with varying alkyl chain lengths and degrees of saturation. These compounds were tested in vitro for activity and selectivity in comparison with PFA and ODG-PFA in cells infected with HCMV, HSV-1 or HIV-1. Antiviral activity was strongly dependent on chain length with alkyl ethers 14-18 carbon atoms long exhibiting the greatest antiviral activity against HCMV and HSV-1. In HIV-infected HT4-6C cells, optimal activity was observed at 18-22 carbon chain lengths. The antiviral activities of 1-octadecyloxypropane-3-PFA and 1-docosyloxypropane-3-PFA were 135- and 338-fold greater than that of PFA in HT4-6C cells infected with HIV-1. This also represents a 2.6-6-fold improvement in antiviral activity over ODG-PFA, the previously reported analog.

Evaluation of a novel lipid prodrug for intraocular drug delivery: effect of acyclovir diphosphate dimyristoylglycerol in a rabbit model with herpes simplex virus-1 retinitis.

BACKGROUND: Acyclovir diphosphate dimyristoylglycerol is a lipid prodrug of acyclovir that forms liposomes and provides substantial activity against herpes simplex virus, acyclovir-resistant strains of herpes simplex virus, and human cytomegalovirus. We therefore tested this promising new drug in a rabbit model of herpes simplex retinitis. METHODS: A total of 22 pigmented rabbits were pretreated with either acyclovir diphosphate dimyristoylglycerol, ganciclovir, acyclovir, or buffer. Retinae then were inoculated with herpes simplex virus-1 or buffer 1 week after the injection of drug. In another experiment we compared the effects of acyclovir diphosphate dimyristoylglycerol and acyclovir diphosphate dioleoylglycerol on the optical clarity of vitreous. RESULTS: Animals injected intravitreally with acyclovir diphosphate dimyristoylglycerol showed retinitis that was less severe than that in animals injected with ganciclovir, acyclovir, and buffer; differences in grading scores of the retinitis between animals injected with acyclovir diphosphate dimyristoylglycerol and those injected with buffer were statistically significant (P = 0.0015). Vitreous and optical media became clear 4 days after acyclovir diphosphate dioleoylglycerol injection compared with 10 days after with acyclovir diphosphate dimyristoylglycerol injections. CONCLUSION: Acyclovir diphosphate dimyristoylglycerol had prolonged antiviral activity against herpes simplex virus-1 retinitis in a rabbit model. This drug delivery system, modified to improve optical clarity, may allow long-acting intravitreal treatment of cytomegalovirus retinitis and other retinal diseases.

Synthesis and antiviral activity of 1-O-octadecyl-2-O-alkyl-sn-glycero-3-foscarnet conjugates in human cytomegalovirus-infected cells.

A series of new lipid prodrugs with the general structure, 1-O-octadecyl-2-X-sn-glycero-3-PFA were synthesized and evaluated for antiviral activity in HCMV-infected human lung fibroblasts (X is -H, -OH or an O-alkyl group of increasing chain length) in order to study structure-activity relationships of PFA lipid prodrugs. The EC50 values for the 2-O-octyl, 2-O-butyl, 2-H, 2-OH, 2-O-methyl and 2-O-ethyl substituted analogs were 1.96, 0.36, 1.0, 0.7, 0.53 and 0.18 microM respectively versus 40 microM for PFA, representing increases in antiviral activity of 20-220 fold. We also synthesized the enantiomer of ODG-PFA, 3-O-octadecyl-sn-glycero-1-PFA, and found that the antiviral activity of both enantiomers as well as the racemate were not significantly different, with EC50 values in the range of 0.67-0.71 microM.

Lipid prodrugs of phosphonoacids: greatly enhanced antiviral activity of 1-O-octadecyl-sn-glycero-3-phosphonoformate in HIV-1, HSV-1 and HCMV-infected cells, in vitro.

Phosphonoformate (PFA) effectively inhibits viral polymerases but is relatively ineffective in virus-infected cells in tissue culture. A lipid prodrug of phosphonoformate was synthesized by coupling the phosphonate residue of phosphonoformate to the sn-3 hydroxyl of 1-O-octadecyl-sn-glycerol. This prodrug, 1-O-octadecyl-sn-glycero-3-phosphonoformate (ODG-PFA), was 93-fold more active than phosphonoformate in cells infected with the AD169 strain of cytomegalovirus (CMV), and 111-147-fold more active in cells infected with three human clinical isolates of CMV. The compound was also 44-fold more active in human immunodeficiency virus-1 (HIV-1) infected cells and 43-fold more active in cells infected with herpes simplex virus (HSV). Studies of the mechanisms of increased antiviral activity indicate that 1-O-octadecyl-sn-glycero-3-[14C]phosphonoformate is taken up more extensively than the free drug by the host MRC-5 human lung fibroblasts. Intracellular enzymes convert 1-O-octadecyl-sn-glycero-3-phosphonoformate to phosphonoformate. This conversion does not occur in the tissue culture medium containing fetal bovine serum (FBS) or in MRC-5-conditioned medium. In view of its greatly increased in vitro potency and selectivity, 1-O-octadecyl-sn-glycero-3-phosphonoformate may be useful in treating viral diseases.

Comparative evaluation of amiodarone-induced phospholipidosis and drug accumulation in Fischer-344 and Sprague-Dawley rats.

Amiodarone (AD) and its major metabolite, desethylamiodarone (desethylAD), are both phospholipogenic. The present study was undertaken to evaluate the comparative susceptibilities of male Fischer-344 and Sprague-Dawley rats to AD-induced phospholipidosis in alveolar macrophages (AMs), liver and kidney tissue and the concomitant accumulation of AD and desethylAD in these cells, tissues and plasma. Rats were administered AD (100 mg/kg/day, p.o.) for 1 week. Plasma concentrations of AD and desethylAD were approximately 4- and 12-fold higher, respectively, in Fischer-344s compared to Sprague-Dawleys 24 h after the last dose. AD and desethylAD levels in AMs were approximately 12- and 25-fold higher, respectively, in Fischer-344s than Sprague-Dawleys. In the liver and kidney, levels of both compounds were also significantly higher in Fischer-344s than Sprague-Dawleys. Ultrastructural features indicative of phospholipidosis were not observed consistently in any tissue except AMs from treated Fischer-344s. AM total phospholipid increased nearly 5-fold in Fischer-344s, while Sprague-Dawleys showed no increase over control. AMs from both strains incubated with 10 microM AD or desethylAD in vitro were not significantly different in their accumulation of the compounds. When incubated with AD or desethylAD, the lysosomal phospholipases A1 partially purified from AMs of both strains were equally sensitive to inhibition as measured by the drug concentration giving 50% inhibition in activity (IC50). The results of this study indicate that at the same administered dose, AD and desethylAD, accumulate to higher tissue levels and are more phospholipogenic in male Fischer-344 rats than in male Sprague-Dawley rats. The basis for the high susceptibility of Fischer-344 rats to AM-induced phospholipidosis is unknown at present but appears not to be related to biochemical or cellular features of the AMs.

Antiviral effect in human cytomegalovirus-infected cells, pharmacokinetics, and intravitreal toxicology in rabbits of acyclovir diphosphate dimyristoylglycerol.

Acyclovir diphosphate dimyristoylglycerol (ACVDP-DG) is a lipid prodrug which is active against ACV-resistant strains of herpes simplex virus because of its intracellular metabolism to ACV monophosphate. In human cytomegalovirus (HCMV)-infected MRC-5 cells, ACVDP-DG was ninefold more active than ACV. When liposomal [8-3H]ACVDP-DG was injected intravitreally at the maximum nontoxic dose of 1 mumol in rabbits, the drug remained above its estimated 90% HCMV-inhibitory concentration for 18 days. Intravitreal ganciclovir persists above its 90% inhibitory concentration for only 1 to 2 days. ACVDP-DG may be useful as a local treatment for HCMV retinitis.

Acyclovir diphosphate dimyristoylglycerol: a phospholipid prodrug with activity against acyclovir-resistant herpes simplex virus.

Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir (9-[(2-hydroxyethoxy)-methyl]guanine, Zovirax) is a growing clinical problem in patients with AIDS and other immunosuppressed states. Most virus isolates resistant to acyclovir are deficient or defective in virally coded thymidine kinase (TK), which converts acyclovir to acyclovir monophosphate in virus-infected cells. To restore acyclovir efficacy, we synthesized acyclovir diphosphate dimyristoylglycerol, an analog of a naturally occurring phospholipid, CDP-diacylglycerol. Its biological activity was tested in WI38 human lung fibroblasts infected with the acyclovir-resistant DM21 strain of HSV, which is TK negative due to an 816-base-pair deletion in the TK coding region. Acyclovir diphosphate dimyristoylglycerol has substantial activity in DM21-infected cells (IC50 = 0.25 microM), whereas acyclovir and acyclovir monophosphate were ineffective (IC50 > 100 microM). Similar results were obtained in TK-altered and TK-deficient strains of HSV-1 and in acyclovir-resistant isolates of HSV-2 obtained from two AIDS patients. The phospholipid prodrug is active by means of TK-independent metabolic pathways that liberate acyclovir monophosphate inside the host cell. Acyclovir phosphates were 56 times greater in WI38 human lung fibroblasts incubated for 24 hr with [8-3H]acyclovir diphosphate dimyristoylglycerol relative to acyclovir. Acyclovir monophosphate added to the culture medium (outside the cell) did not circumvent the acyclovir resistance of the TK-negative DM21 mutant, presumably due to its conversion to acyclovir by phosphatases. Acyclovir diphosphate diacylglycerol prodrugs may be useful in treating TK-deficient mutant and wild-type strains of HSV.

Effect of amiodarone on the phospholipid and lamellar body content of lymphoblasts in vitro and peripheral blood lymphocytes in vivo.

Amiodarone is useful for the treatment of ventricular arrhythmias but has been associated with a significant degree of toxicity especially to lung and liver. The drug produces phospholipid accumulation in multilamella lysosomal inclusions in many tissues due to the ability of amiodarone and desethylamiodarone to inhibit phospholipase A. The adverse effects do not correlate with the plasma levels of amiodarone but relate more closely to the cumulative dose. No clear way of predicting amiodarone toxicity has yet emerged. In this report, normal human lymphoblasts in tissue culture were shown by electron microscopy to have dose-dependent increases in multilamellar inclusions when grown with amiodarone at concentrations which are routinely observed in patients receiving the drug. The content of phospholipid also increased but this parameter was not as sensitive as the number of multilamellar inclusions. Finally, lymphocytes from patients treated with amiodarone were examined by electron microscopy and shown to have increased numbers of multilamellar bodies.

Assay of phospholipases C and D in presence of other lipid hydrolases.

The activity of a phospholipase C or phospholipase D may be assessed by measuring the radioactivity or phosphate released into the aqueous phase of a lipid extract. However, in crude enzyme fractions, this type of analysis may not be possible due to formation of water-soluble metabolites by other enzymatic reactions, as demonstrated here with a crude lysosomal enzyme fraction. In such instances, analysis of both water-soluble and lipid-soluble metabolites, at various times of incubation, may still provide clear identification of phospholipases C or D, even when a variety of lipases and other hydrolases are present.

Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence.

During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.

Methylcholanthrene: a possible pseudosubstrate for adrenocortical 17 alpha-hydroxylase and aryl hydrocarbon hydroxylase.

In cultured bovine adrenocortical cells, loss of 17 alpha-hydroxylase activity was observed after incubation with 3-methylcholanthrene (3-MC). The suppression of 17 alpha-hydroxylase by 3-MC was rapid (50% loss of activity in 10 hr at 1 microM 3-MC), did not exhibit a lag period, and was not affected by cycloheximide. Direct effects of 3-MC on 17 alpha-hydroxylase were observed only at high concentrations, but the concentration for 50% loss of activity was 0.3 microM when 3-MC was added for 24 hr prior to assay of 17 alpha-hydroxylase. High concentrations (to 40 microM) of substrate (progesterone), did not affect the loss of activity due to 3-MC. Loss of 17 alpha-hydroxylase activity was specific; 11 beta-hydroxylase was unaffected and cell growth was unaltered. However, 22-amino-23,24-bisnorchol-5-en-3 beta-ol, an inhibitor of 17 alpha-hydroxylase, partially prevented the loss of 17 alpha-hydroxylase at 1-30 nM. 3-MC is thought to induce cytochrome P-450s via a receptor with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD was without effect on 17 alpha-hydroxylase over the range of 10 nM to 10 microM. Benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, chrysene, and methylphenanthrenes suppressed 17 alpha-hydroxylase at high concentrations (10-50 microM for 50% loss of activity). Some steroids that lack a substituent at position 17 also caused loss of 17 alpha-hydroxylase. Like 17 alpha-hydroxylase, bovine adreno-cortical cell AHH was found to be suppressed by exposure to 3-MC. Compounds that caused loss of 17 alpha-hydroxylase caused loss of AHH, with a similar order of potency and at similar concentrations. Suppression of AHH by 3-MC did not require protein synthesis and was prevented by an inhibitor of enzymatic activity, alpha-naphthoflavone. This implies a degree of similarity of the cytochrome P-450s for 17 alpha-hydroxylase and adrenal AHH, but the activities were shown to be likely due to different enzymes. The suppression of 17 alpha-hydroxylase and AHH by 3-MC appears not to occur by a receptor-mediated mechanism but to be similar to the suppression of 11 beta-hydroxylase and 21-hydroxylase by steroid pseudosubstrates previously observed.

The role of ascorbic acid in the function of the adrenal cortex: studies in adrenocortical cells in culture.

To investigate the role of ascorbic acid in the function of the adrenal cortex, we studied the effects of ascorbate on the regulation of 11 beta-hydroxylase in culture. When primary bovine adrenocortical cells were cultured in a serum-free defined medium in the absence of ACTH, 11 beta-hydroxylase activity declined with a half-time of about 40 h. When 50 microM cortisol, which acts as a pseudosubstrate for 11 beta-hydroxylase, was added to such cultures, 11 beta-hydroxylase activity declined with a half-time of about 6 h. Ascorbate (5 mM) markedly reduced the rate of loss of 11 beta-hydroxylase activity in the presence of cortisol. Previous studies showed that phenolic and sulfoxide antioxidants, which also prevent loss of 11 beta-hydroxylase activity, inhibited the enzyme at concentrations somewhat higher than those required for protective activity. However, ascorbate at concentrations from 10 microM to 5 mM did not inhibit 11 beta-hydroxylase. The same range of ascorbate concentrations added to cells during a 24-h preincubation with cortisol showed increasing prevention of loss of 11 beta-hydroxylase activity. Ascorbate and a lowered concentration of oxygen were synergistic in their protective action. At 2% oxygen, 5 mM ascorbate almost completely prevented loss of 11 beta-hydroxylase activity in the presence of 50 microM cortisol. 11 beta-Hydroxylase activity was reinduced over a period of 5 days in third passage cultures by addition of 1 microM ACTH in defined lipoprotein-free medium. Addition of ascorbate enhanced the reinduction about 2-fold. The action of ascorbate in prevention of pseudosubstrate-mediated loss of activity and in enhancing reinduction of 11 beta-hydroxylase is specific; neither alpha-tocopherol nor selenium prevented loss of 11 beta-hydroxylase in the presence of cortisol or enhanced reinduction of 11 beta-hydroxylase in the presence of ACTH. As an additional test of specificity, it was shown that reinduction of 17-hydroxylase activity was completely unaffected by ascorbate, selenium, or alpha-tocopherol, and addition of cortisol to cultures with high 17-hydroxylase did not result in any loss of enzyme activity. Thus, a major function of ascorbate in the adrenal cortex is as a protective compound for cytochrome.

Hormonal regulation of benzo[a]pyrene metabolism in human adrenocortical cell cultures.

In cultured fetal human adrenocortical cells, metabolism of the carcinogen benzo[a]pyrene was found to be unresponsive to the xenobiotic inducers 3-methylcholanthrene, benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, exposure of cultures to the hormone adrenocorticotropin (ACTH) for 48 hours stimulated benzo[a]pyrene metabolism 3-fold. The major metabolite was the 7,8-diol. Other compounds which stimulate the production of adrenocortical cell cyclic AMP (forskolin and cholera toxin) as well as monobutyryl cyclic AMP also increased benzo[a]pyrene metabolism. Human adrenocortical cells thus provide an unusual example of hormonal regulation of the metabolism of a carcinogen.

Selenium deficiency in cultured adrenocortical cells: restoration of glutathione peroxidase and resistance to hydroperoxides on addition of selenium.

Cultured bovine adrenocortical cells were previously shown to be functionally deficient in selenium and vitamin E when grown in medium supplemented with fetal bovine serum. In the present experiments, the lack of significant bioavailable amounts of selenium in the medium was demonstrated by the finding of only low levels of glutathione peroxidase in the cultured cells (0.008 U/mg protein compared with 0.045 U/mg protein in fresh adrenocortical tissue). When 20 nM selenium as selenite was added to the cultured adrenocortical cells, glutathione peroxidase activity increased continuously over 72 h, with a total increase of about eightfold over this period. Over the same time-course, the highest concentration of cumene hydroperoxide tolerated by the cells without cell death increased progressively from 10 microM to 50 microM. Addition of 1 microM alpha-tocopherol also increased the amount of cumene hydroperoxide tolerated to 50 microM. Cell death was measured by cloning efficiency after removal of cumene hydroperoxide. Addition of either selenium or alpha-tocopherol had little effect on the growth rate of the cells over six passages, even when residual vitamin E was removed from the serum by extraction with ether and residual low molecular weight selenium compounds were removed by dialysis. It is concluded that combined deficiency of selenium and vitamin E, at least in the presence of other components of fetal bovine serum, has little effect on the ability of the cells to survive under normal conditions, as evidenced by continued long-term proliferation. However, the low levels of glutathione peroxidase resulting from selenium deficiency cause an increase susceptibility to peroxide-mediated toxicity. The combined deficiency of selenium and vitamin E impairs the ability of cells to survive under adverse conditions, as well as altering mitochondrial functions, as previously demonstrated.

Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11 beta-hydroxylase activity in cultured bovine adrenocortical cells.

When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11 beta-hydroxylase (cytochrome P-45011 beta), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11 beta-hydroxylase against loss of enzymatic activity in the presence of pseudosubstrates:BHA (butylated hydroxyanisole,2(3)-tert-butyl-4-methoxyphenol), dimethyl sulfoxide (DMSO), and metyrapone. The present experiments examine the protective effects of several phenolic analogs of BHA in this system, and compare their activities to that of DMSO and metyrapone. When a variety of analogs of BHA were tested for their abilities to prevent loss of 11 beta-hydroxylase activity in cultured adrenocortical cells incubated with 50 microM cortisol for 24 hr, phenol itself was found to be about equipotent with BHA. Addition of methyl, methoxy and benzyl groups to phenol did not diminish protective activity of the compound, but addition of one and particularly two tert-butyl groups greatly diminished activity. Thus, BHT(2,6-di-t-butyl-4-methylphenol) was inactive, in contrast to BHA. The hydroxy group of phenol was essential since benzene and fluorobenzene were inactive. Compounds with multiple hydroxyl groups were not as active as phenol itself, with the exception of catechol. No products of phenol formed during incubations of cells with cortisol were detected by high performance liquid chromatography. Estimated EC50 values for protection of 11 beta-hydroxylase by phenols were about 100 microM, whereas the EC50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarithmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11 beta-hydroxylase, phenols and DMSO were tested for prevention of loss of 11 beta-hydroxylase activity at three different oxygen concentrations (2, 5, and 19% O2). Lowering the oxygen concentration itself resulted in a small diminution of the loss of 11 beta-hydroxylase. Phenols and dimethyl sulfoxide were more effective at low oxygen and less effective in air. Because the cytochrome P-450 inhibitor metyrapone was found previously to be very effective in protecting 11 beta-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11 beta-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11 beta-hydroxylase, but at concentrations that ranged from 4- to greater than 100-fold higher than those required for protection.(ABSTRACT TRUNCATED AT 400 WORDS)