Effects of Inorganic Lead on Isolated Rat Brain Mitochondrial Respiration

Abstract

In the ndult rat, fed 2, the activity of jejunal sucreae shows a diurnal rhythm with a peak during the dark period. Ontogenitally this enzyme doe^ not appear until approximately the 16th postnatal day; its activity then rises rapidly and re ache^ adult levels by the 35th day. Our aim was to determine whether the diurnal rhythm is present from the outset or appeers only after sucrase has attaine6 adult levels. Rat pups were raised in litters of 9 with a li~htldark cycle of l?.h. Solid food and water were available to mothers and pups at all times and mothers were normally removed on the ?let postnatal day. Four litters were studied during the 24h period beginninu at Dam on the 19th day. One pup from each litter was removed for assay every 3h. Jejunal sucrase shoved considerable variation between pups, but there was no discernable pattern with time. In contrast, when the experiment was repeated with pups aaed 22 days, there was a distinct peak in sucrase activity during the early part of the dnrk period. In order to determine whether the rhythm la cued by the procesm of weaning. two litters were raised on a feeding schedule which ensured that maternal milk was the only food available to the pupa. h%en studied during the 24h period beginning on the 22nd day, the jejunal sucrase activity of these pups exhibited no rhythm. It is concluded that some component of solid food is responsible for the usual appearance of the diurnal rhythm of this enzyme. --0xid;tive'phosphorylation was measured polaribraphically before and after addina lead acetate to cerebral and cerebellar mitochondria isolatei from two week old and adult rats. With glutamate and malate as substrates. lead acetate (0.25 mM) produced a transient increase in respiration followed by complete inhibition of ADP-dependent respiration. The inhibition was not relieved by dinitrophenol or by freezing and thawing the mitochondria. NAOH-supported respiration, in frozen and thawed mitochondria, was not affected by these low concentrations suggesting that lead acts on substrate transport or dehydrogenases. With succinate as substrate, inorganic lead produced an increased respiratory rate, approaching State 3, and no respiratory inhibition. The enhanced respiration was inhibited by oligomycin. There were no differences in lead effects associated with age, two weeks vs adult, or with brain region. cerebral vs cerebellar. We conclude that there is an energydependent transport of inorganic lead in brain mitochondria. Low concentrations of lead inhibit oxidative phosphorylation with NAD-linked substrates but not with succinate. These in vitro lead effects are similar to those seen in cerebellar mitochondria from lead-treated imnature rats (Holtzman and Hsu. Pediat. Res. 10:70-75. 1976). However, this in-vitro mitochondrial effect does not show the age and re-pecificity seen in lead encephalopathy in the rat and the human. 14 A M M O N I A P R O D U C T I O N BY T H E P R E G N A N T S H E E

Effect of Inorganic Lead on Rat Brain Mitochondrial Respiration and Energy Production

“…The concentration range of PbAc required to produce an inhibition of respiration was an order of magnitude (10 times) less than the lowest amounts used in previous studies. Holtzman et al (1978) found that at concentrations below 50 p~, PbAc did not appear to inhibit brain mitochondrial respiration. This may have been caused by the presence of Pi and 0.2 mM-EDTA in their incubation medium at the time of Pb2+ addition.…”

“…It was proposed by Holtzman et al (1978) that lead inhibits a step in the citric acid cycle substrate oxidation prior to NADH dehydrogenase, because NAD-linked substrates are more sensitive to the effects of lead. This appears to be supported from data obtained in the present experiments with selected substrates.…”

Ruthenium Red Inhibition of In Vitro Lead Effects on Brain Mitochondrial Respiration

“…Inhibition of the Pb-induced increase in respiration by Ruthenium Red supports the hypothesis that Pb2+ and Ca2+ share carrier or binding sites in brain mitochondria (Holtzman et al, 1978). La3+ inhibition of Pb2+ uptake suggests a similar analogy of Pb2+ and Caz+ uptake in rat heart mitochondria (Scott et al, 1971).…”

“…The effects of the concentrations of Pb(Ac), shown in Fig. 1 on cerebral mitochondrial respiration were the same as previously described (Holtzman et al, 1978). As shown in Fig.…”

“…Mitochondrial isolates were frozen for subsequent protein determinations (Lowry et al, 1951). The quality and sources of all chemicals are given in our previous publication (Holtzman et al, 1978). Ruthenium Red was obtained from Sigma Chemical Co., St. Louis, Missouri.…”