The antioxidant β-carotene prevents covalent cross-linking between cholesterol side-chain cleavage cytochrome P450 and its electron donor, adrenodoxin, in bovine luteal cells

Abstract

Steroid hormones are an important class of hormones synthesized from cholesterol by a number of endocrine organs; including ovaries, placenta, testes and adrenal glands. The first and rate-limiting step in steroidogenesis is the cleavage of the side-chain of the cholesterol molecule, catalysed by a cytochrome P450 enzyme, cholesterol side-chain cleavage enzyme. This enzyme, as with other P450 enzymes, produces oxygen radicals. Oxygen free radicals can cause deleterious effects such as cross-linking and aggregation of proteins. Cells can protect against such damage with the use of antioxidants. The corpus luteum, or ‘yellow body’, of the ovary is very steroidogenic and is exceedingly rich in the yellow antioxidant, β-carotene. The corpus luteum produces the steroid hormone progesterone that is needed to support pregnancy. Here we have shown that by depleting, or conversely repleting, luteal cells of their β-carotene content in vitro that P450 side-chain cleavage enzyme became covalently non-disulfide cross-linked to its electron donor, adrenodoxin, and hence inactivated. Bovine luteal cells were cultured in 10% fetal calf serum with of without additional treatments for up to 72 h. Under control conditions the cellular levels of β-carotene and α-tocopherol fell by 50% within 24 h and remained low. P450 side-chain cleavage enzyme become non-disulfide covalently cross-linked to its electron donor, adrenodoxin, as determined by Western immunoblotting ( N = 18). Aminoglutethamide inhibited this cross-linking. The addition of β-carotene at levels found in bovine serum, but not α-tocopherol or ascorbic acid, inhibited the degree of the cross-linking. These observations thus offer a good explanation for the very high levels of β-carotene found in bovine corpora lutea, namely that β-carotene protects cholesterol side-chain cleavage P450 against damage from its own oxygen free radicals. They may also explain many observations showing that antioxidant vitamins support the synthesis of steroid hormones, particularly those involved in the regulation of reproduction.