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HAEM SYNTHESIS IN CULTURED HUMAN AND ANIMAL CELLS FOR THE ASSESSMENT OF TOXICITY

التبويبات الأساسية

Abdul-Mawla  M.S. SABBAGH

 

Univ.

Leeds

Spec.

Chemical Pathology

Deg.

Year

#Pages

Ph.D.

1993

177

 

Methods were established for the examination and estimation of all the biosynthetic intermediates of haem  using RP-HPLC.

Optimum conditions for the growth, differentiation and synthesis of haem from exogenous δ-ALA were established for human (K562 and HepG2) and mouse (707B) cell lines. Induction of the K562 cells with 50 µmo1/1 haemin, and the 707B cells with 1.5 % DMSO for 96 h increased cellular haem content 4-fold. All three cell types accumulated large amounts of tetrapyrroles when incubated with 500 µmol/l -ALA. The main accumulated product, after 4 days culture, was PP, up to 735.2 ± 49.7 (K562), 1050.5 ± 80.5 (707B), 590.7 ± 30.0 (HepG2), and 360.0 ± 22.0 (Bone Marrow MNC) µmol/ 106 cells.

Studies of uptake and subcellular distribution of AL, Cd and Pb showed that in serum-free media all three cell lines exhibited a non-saturatable uptake of the metals against a concentration gradient. Al and Cd uptake was completely prevented in the presence of 10% FCS (vol/vol). The majority of metals taken up was located in the nuclear fraction (>40%) with small but significant (p<0.001) amounts in the mitochondrial (>7%) and cytosolic (>5%) fractions.

Porphyrinogenicity of metals (Al, Cd and Pb) and organic substances (HCB, lindane and DDC) have been studied in the cell cultures and the methodology resulted from these techniques were applied to human bone marrow samples.

The amount of PP accumulated by 707B, K562 and human bone marrow mononuclear cells increased with increasing Al (up to 10 µmol/1) and Cd (up to 25 µmol/1) concentrations as cellular haem declined. Al at 10 µmol/l and Cd at 25 µmol/1 increased PP accumulation 2-fold. On the other hand, when these cells were challenged with 25 µmol/l Pb, they exhibited 3-fold, 4-fold and' approx 2-fold increase in PP, Copro and c8 - c5 porphyrins, respectively. The increase in PP, which occurred mainly in the cells was about equal to the decrease in cellular haem. Copro accumulated mainly in the media.

Pretreatment of HepG2 cells with either HCB of lindane resulted in the accumulation of mainly Uro and heptacarboxyporphyrin in a dose-dependent manner which coincided with an equivalent decrease in PP. At 10 µmol/l both chemicals increased c8 -c5 porphyrin accumulation 10-fold, over control values.

The addition of up to 10 µmol/l DDC to the 707B of HepG2 cells produced a 3-fold rise In PP accumulation, which coincided with an equivalent decrease in cellular haem.

It was concluded that, porphyrin patterns may serve as a guide to which enzyme of haem. biosynthesis is inhibited in cell cultures, and that the K562, 707B and HepG2 cell lines are valid model systems for studying the porphyrinogenicity of metals and organic substances.