Reduction in porphyrin excretion as a sensitive indicator of lead toxicity in primary cultures of adult rat hepatocytes

Alterations of specific metabolic pathways can be used as sensitive indicators of toxicity by chemicals and can give valuable information on the mechanism(s) involved. Short-term effects of lead on hepatic haem biosynthesis were studied in an in vitro system. Primary cultures of adult rat hepatocytes were exposed for 24–48 hr to lead (0.024–3.6 m m ), and excreted and intracellular porphyrins were measured in untreated and lead-treated cultures. Cytotoxicity, as estimated by enzyme leakage, and morphological alterations were also evaluated. Control hepatocytes produced porphyrins at a rate of 387 pmol/mg cellular protein/day. Most of the released and intracellular porphyrins were protoporphyrins, although uro- and coproporphyrins were also detected in lower amounts. After 24 hr of exposure to 0.1–3.6 m m Pb 2+ , excreted porphyrins decreased by 24–92% and intracellular porphyrins by 36–60%, while 48 hr of exposure to 0.024–3.6 m m Pb 2+ caused a progressive reduction of 77–97% in porphyrin excretion and of 49–67% in intracellular porphyrins. Lead exposure also produced a differential decrease of proto-, copro- and uro-porphyrin excretion. These lead effects can be explained mainly by inhibition of the enzyme 5-aminolaevulinate dehydratase, resulting in a decreased monopyrrole supply for porphyrin biosynthesis, and probably by inhibition of the enzyme uroporphyrinogen decar☐ylase. Morphological alterations and enzyme leakage were detected only after 24 hr of exposure to 2.4 m m and 48 hr of exposure to 3.6 m m Pb 2+ , respectively. The results show that changes in porphyrin production, and particularly in their excretion, in cultured rat hepatocytes are useful indicators of lead toxicity, since they are more sensitive than enzyme leakage and can give preliminary information on the enzyme(s) that could be affected. They also suggest the potential benefits of the use of this method for the evaluation of compounds that alter haem biosynthesis.