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Effects of Lead on Haem Biosynthesis During Erythroid Differentiation in Vitro

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Effects of Lead on Haem Biosynthesis During Erythroid Differentiation in Vitro Toxic. in Vitro Vol. 4, No. 6, pp. 763-769. 1990 0887-2333/90 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright ~ 1990 Pergamon Press plc EFFECTS OF LEAD ON HAEM BIOSYNTHESIS DURING ERYTHROID DIFFERENTIATION IN VITRO W. W. Ku, D. SLOWIEJKO, L. L. BESTERVELT, M. R. BUROKERand W. N. PIPER* *Toxicology Program, School of Public Health, and Department of Pharmacology, Medical School, University of Michigan, Ann Arbor, MI 48109, USA (Received 26 September 1989; revisions received 16 February 1990) Al~tract--Murine erythroleukaemia cells (MELC) are erythroid precursor cells that undergo erythroid differentiation in the presence of the inducer hexamethylene bisacetamide (HMBA). The effects of lead on haem biosynthesis in MELC following HMBA-induced differentiation were studied. MELC were induced with HMBA in the presence of 20, 40 and 80/aM-lead acetate and cell density, haem content, incorporation of ~4C-labelled6-aminolaevulinic acid (ALA) into haem, and the activities of the enzymes 6-aminolaevulinic acid dehydratase (ALA-D), uroporphyrinogen I synthetase (URO-S) and ferrochelatase (FERRO) were determined. MELC exposed to 80/~M-lead showed significant erythroid hypoplasia (40-50%) and a significant decrease (30-50%) in haem content at 2, 4 and 6 days after induction in comparison with the controls. Significant inhibition of ALA-D, the most sensitive index, was noted at 20 ~UM-lead,and at 80/~M-lead ALA-D activity was decreased by 60-80°/, in comparison with the controls. URO-S and FERRO showed significant decreases of 34% and 50%, respectively, at 80/zM-lead. A decrease of 50% in the incorporation of [~4C]ALA into haem at 80/~M-lead indicated an impairment in haem synthesis. The results suggest that the impairment of haem formation by lead is coincident with the production of severe erythroid hypoplasia. INTRODUCTION The haem biosynthetic pathway in erythroid tissue Lead has long been known to exert toxic effects on is a well known target for the toxic effects of lead the erythropoietic system that are associated with the (Bottomley and Muller-Eberhard, 1988). Lead has development of anaemia. There is little information been shown to inhibit virtually all the enzymes of available concerning the regulation of haem bio- the pathway. 6-Aminolaevulinic acid dehydratase synthesis in the bone marrow associated with the (ALA-D) is most sensitive to lead in vitro and in vivo, development of anaemia following exposure to lead. followed by ferrochelatase (FERRO), copropor- Most previous studies concerning the aetiology of phyrinogen oxidase and porphobilinogen deaminase drug/toxicant-associated anaemia and its relationship (PBGase, uroporphyrinogen I synthetase), respect- to the biosynthesis of haem have been conducted with ively. Very high lead concentrations inhibit uropor- the mature red blood cell, which is not actively phyrinogen decarboxylase and di-aminolaevulinic engaged in haem synthesis. Therefore, more relevant acid synthase (Bottomley, 1977). information is likely to be obtained by focusing study As a result of the complex cellular heterogeneity of on the bone marrow rather than the red blood cell. bone marrow in vivo, controlled study of this cellular Furthermore, controlled study of the role of various target in the whole animal is rather difficult. There- physiological (hormonal, nutritional) factors that fore, an in vitro system was used to study lead regulate haem synthesis and/or influence the develop- interactions and regulation of haem synthesis in ment of lead- or drug-associated anaemias is lacking. erythroid precursor cells. The relative ease with which It is known that lead is preferentially deposited in conditions can be manipulated in vitro makes it bone marrow, and its concentration may reach 50 possible to study the role of various hormonal and times that in blood (Albahary, 1972). Consequently, nutritional factors that may regulate haem synthesis bone marrow cells, particularly premature erythroid and/or influence the development of lead- or drug- precursor cells, are most susceptible to the toxic associated anaemias. effects of lead which can result in an anaemia charac- Murine erythroleukaemia cells (MELC) are a terized by erythroid hypoplasia (Sassa, 1978). continuous cell line of erythroid precursor cells which, in the presence of certain chemicals such as dimethylsulphoxide and hexamethylene bisacetamide *To whom correspondence should be addressed. (HMBA), undergo changes similar to the normal Abbreviations: ALA = 6-aminolaevulinic acid; ALA-D = maturation of red blood cells (Friend et al., 1971; 6-aminolaevulinic acid dehydratase; BPb = blood lead Reuben et al., 1976). These changes include alter- concentration; DTT = dithiothreitol; FERRO = ferro- ations in morphology, sequential induction of chelatase, HMBA= hexamethylene bisacetamide; MELC = murine erythroleukaemia cells; MEM = enzymes in the haem biosynthetic pathway with the minimum essential medium; PBG =porphobilinogen; production of haem and haemoglobin, expression of PBGase = porphobilinogen deaminase; PBS = phos- carbonic anhydrase activity, and the appearance of phate buffered saline; URO-S=uroporphyrinogen ! spectrin and red blood cell surface antigens (Marks synthetase. and Rifkind, 1978). It is anticipated that MELC will 763 764 W.W. Ku et al. be a useful in citro model to study drug/toxicant Spectrophotofluorometer (American Instrument Co. interactions during erythropoiesis. Inc., Silver Spring, MD, USA). In the studies described here we investigated the Subcellular fractionation. At the indicated times, effects of lead on haem biosynthesis in MELC follow- MELC were collected by centrifugation at 100g ing HMBA-induced erythroid differentiation. The for 10min and rinsed with PBS. The washed cell studies were designed to assess the ability of lead to pellets (from control and lead-exposed MELC) were (I) impair haem formation through a disruption in weighed and resuspended in three volumes of ice-cold the normal maturation process and/or perturbation distilled deionized water to lyse cells. These suspen- of selected enzymes m the haem biosynthetic sions were homogenized manually with 15 strokes in pathway, and (2) to produce a state of erythroid a glass-teflon homogenizer and then adjusted to hypoplasia in differentiating MELC). pH 7.8 by the addition of six volumes of 50 mM-Tris- HCI (pH 8.0). The homogenate was centrifuged at 800g for 15min to remove cellular debris. The MATERIALS AND METHODS resulting supernatant was centrifuged at 9000g for 20 min, yielding a mitochondrial pellet. This mito- Cell culture. The murine erythroleukaemia cell line, chondrial pellet, washed and resuspended in 50 mM- clone 745A, derived from the DBA/2 mouse, was Tris-HCl (pH 7.4), served as the enzyme source. The obtained from the NIGMS Human Genetic Mutant resulting 9000-g supernatant was centrifuged at Cell Repository, Institute for Medical Research, 100,000g for 60min to form the cytosolic fraction (Camden, N J, USA). Cells were seeded at a final and the microsomal pellet. The cytosolic fraction density of 5 x 105cells/ml in spinner flasks. Spinner served as the enzyme source. Protein content was cultures were maintained in an incubator at 37~C in determined by the method of Lowry et al. (1951) medium consisting of Iscove's modified Dulbecco's using bovine serum albumin as standard. minimum essential medium (MEM); Boehringer Activities of haem biosynthetic enzymes. The Mannheim Biochemicals, Indianapolis, IN, USA) activity of ALA-D was measured in MELC cytosol supplemented with 5% (v/v) defined bovine calf by the method of Gibson et al. (1955). The product, serum (Hyclone Laboratories Inc., Logan, UT, porphobilinogen (PBG), was measured spectrophoto- USA). Induction of differentiation was achieved by metrically (553 nm) after reaction with Ehrlich's addition of a stock solution (250mM) of HMBA reagent (Mauzerall and Granick, 1956). Spectro- (Sigma Chemical Co.. St Louis, MO, USA) to a final photometric determination of PBG was carried out medium concentration of 3 raM. To assess the effects on an SLM Aminco DW-2000 UV-VIS Spectro- of lead, MELC were induced to differentiate in the photometer (SLM Instruments Inc., Urbana, IL, continuous presence of 0 (control), 20, 40 or 80 p~- USA) using a millimolar extinction coefficient of 61. lead by appropriate freshly prepared dilutions in Cytosolic uroporphyrinogen I synthetase (URO-S) medium of a stock lead acetate solution in distilled was measured by a modification (Sassa et al., 1974) deionized water (10 mM). Lead concentrations were of the method of Strand et al. (1972). The cytosolic chosen on the basis of solubility limits in culture preparation was incubated with 100/~M-PBG (Por- medium and cell viability (trypan blue exclusion) phyrin Products, Logan, UT, USA) in 50 mM-Tris- during exposure periods. The actual lead concen- HCI-50 mr,l-KC1 (pH 7.8) at 37 C for 45 min. The trations in medium were 19.5, 38.7 and 77.2pM as reaction was terminated by cooling tubes on ice and determined by atomic absorption spectrophotometry immediately adding ethyl acetate-acetic acid (2: 1, (Chemical Pathology, Department of Pathology, v/v). Then 1.0 N-HCI was added and the mixture Medical School, University of Michigan, MI, USA). was shaken vigorously. The phases were allowed to The lead concentration in control medium was below separate and stand for 30 min in room light. The the limits of detection. Under all conditions, cell porphyrin fluorescence in the lower aqueous phase viability was >90%. Cultures
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