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

Peter J Hornsby, Kathy A. Aldern, Sandra E. Harris

Research output: Contribution to journalArticle

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Abstract

When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11β-hydroxylase (cytochrome P-45011β), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11β-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 11β-hydroxylase activity in cultured adrenocortical cells incubated with 50 μM 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 11β-hydroxylase by phenols were about 100 μM, whereas the ec50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarothmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11β-hydroxylase, phenols and DMSO were tested for prevention of loss of 11β-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 11β-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 11β-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11β-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11β-hydroxylase, but at concentrations that ranged from 4- to >100-fold higher than those required for protection. In contrast, for metyrapone, the ec50 values for protection and inhibition were very similar. These results indicate that it is unlikely that phenols act simply as inhibitors but may need to bind close to the active site of the enzyme. The observed synergism with lowered oxygen suggests an involvement, in the loss of 11β-hydroxylase activity, of oxygen-centered radicals that may be reactants for protective phenols.

Original languageEnglish (US)
Pages (from-to)865-872
Number of pages8
JournalBiochemical Pharmacology
Volume34
Issue number6
DOIs
StatePublished - Mar 15 1985
Externally publishedYes

Fingerprint

Butylated Hydroxyanisole
Phenols
Mixed Function Oxygenases
Dimethyl Sulfoxide
Metyrapone
Phenol
phenol 2-monooxygenase
Oxygen
Hydrocortisone
Butylated Hydroxytoluene
Fluorobenzenes
Steroid 11-beta-Hydroxylase
Enzymes
Cytochromes
Benzene
High performance liquid chromatography
Hydroxyl Radical
Cultured Cells
Reactive Oxygen Species
Catalytic Domain

ASJC Scopus subject areas

  • Pharmacology

Cite this

Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11β-hydroxylase activity in cultured bovine adrenocortical cells. / Hornsby, Peter J; Aldern, Kathy A.; Harris, Sandra E.

In: Biochemical Pharmacology, Vol. 34, No. 6, 15.03.1985, p. 865-872.

Research output: Contribution to journalArticle

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abstract = "When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11β-hydroxylase (cytochrome P-45011β), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11β-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 11β-hydroxylase activity in cultured adrenocortical cells incubated with 50 μM 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 11β-hydroxylase by phenols were about 100 μM, whereas the ec50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarothmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11β-hydroxylase, phenols and DMSO were tested for prevention of loss of 11β-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 11β-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 11β-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11β-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11β-hydroxylase, but at concentrations that ranged from 4- to >100-fold higher than those required for protection. In contrast, for metyrapone, the ec50 values for protection and inhibition were very similar. These results indicate that it is unlikely that phenols act simply as inhibitors but may need to bind close to the active site of the enzyme. The observed synergism with lowered oxygen suggests an involvement, in the loss of 11β-hydroxylase activity, of oxygen-centered radicals that may be reactants for protective phenols.",
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N2 - When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11β-hydroxylase (cytochrome P-45011β), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11β-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 11β-hydroxylase activity in cultured adrenocortical cells incubated with 50 μM 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 11β-hydroxylase by phenols were about 100 μM, whereas the ec50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarothmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11β-hydroxylase, phenols and DMSO were tested for prevention of loss of 11β-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 11β-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 11β-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11β-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11β-hydroxylase, but at concentrations that ranged from 4- to >100-fold higher than those required for protection. In contrast, for metyrapone, the ec50 values for protection and inhibition were very similar. These results indicate that it is unlikely that phenols act simply as inhibitors but may need to bind close to the active site of the enzyme. The observed synergism with lowered oxygen suggests an involvement, in the loss of 11β-hydroxylase activity, of oxygen-centered radicals that may be reactants for protective phenols.

AB - When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11β-hydroxylase (cytochrome P-45011β), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11β-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 11β-hydroxylase activity in cultured adrenocortical cells incubated with 50 μM 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 11β-hydroxylase by phenols were about 100 μM, whereas the ec50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarothmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11β-hydroxylase, phenols and DMSO were tested for prevention of loss of 11β-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 11β-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 11β-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11β-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11β-hydroxylase, but at concentrations that ranged from 4- to >100-fold higher than those required for protection. In contrast, for metyrapone, the ec50 values for protection and inhibition were very similar. These results indicate that it is unlikely that phenols act simply as inhibitors but may need to bind close to the active site of the enzyme. The observed synergism with lowered oxygen suggests an involvement, in the loss of 11β-hydroxylase activity, of oxygen-centered radicals that may be reactants for protective phenols.

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