Estimation of creatine phosphokinase and hydroxyproline in the developing limb: Its use in evaluating the effect of teratogens on myogenesis and chondrogenesis

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Abstract
Forelimbs of mouse fetuses were examined for tissue‐specific, drug‐induced alterations in their biochemical composition. The activity of the enzyme creatine phosphokinase (CPK; to estimate myogenesis) and the content of hydroxyproline (HP; to estimate chondrogenesis) were compared in homogenates of control and treated mouse‐fetus forelimbs on day 14 of gestation. In addition, the content of DNA, RNA, and protein was also measured. Injection of 6‐aminonicotinamide (6‐AN) (15 mg/kg) on day 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of HP:CPK was unaffected by 6‐AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a significant reduction in day 14 forelimb HP and RNA content, without altering CPK, DNA, or protein; thus, the HP:CPK ratio was decreased. These results indicated that (1) 6‐AN nonspecifically retards growth and cytodifferentiation in limbs; (2) retinoic acid inhibits synthesis of collagen and RNA; (3) retinoic acid has a differential effect upon chondrogenic and myogenic tissues of the limb, and (4) the comparison of HP content and CPK activity in tissue homogenates is an acceptable method of evaluating teratogenic compounds for selective effects.