Fd Chem. Toxic. Vol. 24, No. 6/7. pp. 801-802, 1986 0278-6915/86 $3.00+0.00 Printed in Great Britain Pergamon Journals Ltd

PERMEABILITY OF ISOLATED HEPATOCYTES A N D IMPERMEABILITY OF CULTURED HEPATOCYTES

AND LIVER SLICES TO EDTA

D. P. HUE*, D. BEALES, K. L. GRIFFITH and A. E. M. MCLEAN Laboratory of Toxicology, Department of Clinical Pharmacology, University College London,

London WC1E 6JF, England

Introduct ion

The calcium salt of ethylenediaminetetraacetic acid (EDTA) at a concentration of 4 mM can prevent the toxicity exhibited by paracetamol in suspensions of isolated hepatocytes (Beales, HUe & McLean, 1985) and one of its benefits is a reduction in the degree of lipid peroxidation. However, we report here on the inability of C a E D T A to protect against paracetamol injury in other in vitro systems and relate this to the impermeability of these models to CaEDTA.

Exper imenta l

Male Wistar rats (body weight 180-250g) were given 0.1% phenobarbital in their drinking-water for 5 days. On the day before they were killed the animals were given an oral dose of 20 mg vitamin E. Isolated hepatocytes were prepared by collagenase perfusion of the liver and were incubated as described by Devalia, Ogilvie & McLean (1982). Liver slices were prepared as previously described (McLean & Nuttall, 1978). Primary cultures of hepatocytes were prepared as described by Hue, Griffith & McLean (1985).

The period of exposure to paracetamol (10mM) was dependent upon the system under investigation, suspensions of hepatocytes being exposed for 1 hour,

*Present address: Department of Immunology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE.

liver slices for 2 hours and primary Cultures for 4 hours. The latter were also treated with 50/~M D,L-buthionine-[S,R]-sulphoximine (BSO) for 2 hours prior to and during the paracetamol exposure period. BSO is known to be a potent inhibitor of glutathione synthesis (Griffith & Meister, 1979). The total length of these incubations varied from 4 to 24 hours (see Tables 1 & 2). When C a E D T A was included, it was present at a final concentration of 4 mM for the entire incubation time.

For the studies on [~4C]EDTA uptake, 1/~Ci [14C]EDTA in a final concentration of 4mM C a E D T A was used. Uptake of the label was mea- sured over 30 minutes in all three models. The volumes of extracellular and intracellular space in the cells and slices were estimated using tritiated water and [methoxy-14C]inulin.

Resul ts and Discuss ion

C a E D T A readily protects against paracetamol in- jury in suspensions of isolated hepatocytes (Beales et al. 1985). However, we have found that in primary cultures (Table 1) and in liver slices (Table 2) the chelate has no protective effect.

We propose that this inability to block par- acetamol toxicity is due to these more ordered struc- tures being impermeable to the chelate. This in turn suggests that E D T A must be present in the cytosol in order to exert its protective effect. As seen in Table 3, the accumulation of [~4C]EDTA, measured

Table 1. The effect of CaEDTA on lactate dehydrogenase (LDH) leakage in BSO- and paracetamol-treated primary cultures of rat hepatocytes

LDH leakage (% of total) at:

Treatment 6 hr 24 hr

Control 7.1 + 2.2 30.4 _ 10.2 BSO + paracetamol 2.1 + 2.9 56.9 _+ 6.0 CaEDTA, BSO + paracetamol 2.0 + 1.7 56.5 + 9.0

BSO = n,L-Buthionine-[S,R]-sulphoximine

Table 2. The effect of CaEDTA on lactate dehydrogenase (LDH) leakage in paracetamol-treated rat-liver slices

LDH leakage (% of total) at:

Treatment 4 hr 6 hr

Control 2.9 + 1.8 5.4 _+ 3.6 Paracetamol 11.9 _+ 7.1 32.2 + 11.8 Paracetamol + CaEDTA 12.5 _+ 5.6 31.8 + 15.7

801

802 D.P . HUE et al.

Table 3. Accumulation of [14c]EDTA by various in vitro rat liver models during a 30-rain incubation with 4 mM Ca[~4C]EDTA

Concn of EDTA in:

System ICS (mu) ICS/ECS (%)

Suspensions 1.80 + 0.06 45.0 Primary cultures 0.18 + 0.06 4.5 Liver slices 0.47 + 0.47 11.7

ICS = Intracellular space ECS = Extracellular space

after 30 minutes, is rapid in suspensions of isolated hepatocytes bu t negligible in the o ther systems.

REFERENCES

Beales D., Hue D. P. & McLean A. E. M. (1985). Lipid peroxidation, protein synthesis and protection by CaE-

DTA in paracetamol injury to isolated hepatocytes. Bio- chem. Pharmac. 34, 19.

Devalia J. L., Ogilvie R. C. & McLean A. E. M. (1982). Dissociation of cell death from covalent binding of par- acetamol by flavones in a hepatocyte system. Biochem. Pharmac. 31, 3745.

Griffith O. W. & Meister A. (1979). Potent and specific inhibition of glutathione synthesis by DL-buthionine-S,R- sulphoximine. J. biol. Chem. 254, 7558.

Hue D. P., Griffith K. L. & McLean A. E. M. (1985). Hepatocytes in primary culture become susceptible to paracetamol injury after depletion of glutathione using DL-buthionine-S,R-sulphoximine. Biochem. Pharmac. 34, 4341.

McLean A. E. M. & Nuttall L. (1978). An in vitro model of liver injury using paracetamol treatment of liver slices and prevention of injury by some antioxidants. Biochem. Pharmac. 27, 425.