ELSEVIER European Journal of Pharmacology Environmental Toxicology and Pharmacology Section 293 (1995) 335-339

environmental toxicology and pharmacology

Down-regulation of hepatic peripheral-type benzodiazepine receptors caused by acute lead intoxication

e ra Fonia a, Ronit Weizman b, Eliyahu Zisman c, Ruth Ashkenazi d, Moshe Gavish a,e,* • Department of Pharmacology. The Bruce Rappaport Faculty ofMedicinc, Technion-lsrael Institute of Technology. Haifa. Israel

b Tel Aviv Community Mental Health Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel c Department of Anesthesiology. Rambam Medical Center, Half a, Israel

d Institute for Occupational Health and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel c Rappaport Family Institute for Research in the Medical Sciences. Haifa. Israel

Received 16 March 1995; accepted 30 June 1995

Abstract

In the present study we investigated the influence of acute lead poisoning upon the expression of benzodiazepine receptors. In addition, we examined if administration of PK I i 195, an isoquinoline carboxamide derivative, to lead-poisoned rats could modulate the changes in receptor binding properties achieved by lead alone. Lead poisoning was ascertained by determination of urine &aminolevu- linic acid levels and lead levels in rat livers. Scatchard analysis of saturation curves of [3H]PK I 1195 binding to liver membranes of rats treated with lead alone or with both lead and PK I 1195 showed an approximately two-fold decrease in receptor density in comparison with control groups. Peripheral benzodiazepine receptor density in the kidneys and adrenals of poisoned rats was not changed by lead intoxication per se or by coadministration of PK 11195. Scatchard analysis of saturation curves of [3HIRe 15-1788 binding in rat cerebral cortex tissue showed no difference in the receptor density between the various groups. The K, values of all organs were in the nanomolar range (1-4 nM). We conclude that PK 11195 is not a protective agent of hepatic peripheral benzodiazepine receptors in lead intoxication. Moreover, it causes over-accumulation of lead in hepatocytes in an unknown mechanism of action.

Keywords: Benzodiazepine receptor, peripheral; Benzodiazepine receptor, central; PK I 1195; Re 15-1788; Lead poisoning; Liver

1, Introduction

Acute lead poisoning is manifested by hepatic damage and hemolysis. There are also signs and symptoms refer- able to the nervous, hematological, renal, gastrointestinal, and cardiovascular systems (Cullen et ai., 1983; Lockitch, 1993). The pharmacological mechanism of action of lead includes its combination with sulfhydryl groups of biologi- cal compounds (Putnam, 1986). Lead interferes with the biosynthesis of heine at the enzymatic step where 8- aminolevulinic acid is converted by &aminolevulinic acid dehydrase into porphobilinogen, and at the last biosyn- thetic step of this pathway, where an iron atom is inserted

• Corresponding author. At: Department of Pharmacology, The BnJc¢ Rappaport Faculty of Medicine, Technion-lsra¢l Institute of Technology, P.O.B. 9649, 31096 Haifa, Israel. Tel.: 972.-4-295275; Fax: 972~-532102.

Elsevier Science B.V. SSD! 0 9 2 6 - 6 9 1 7 ( 9 5 ) 0 0 0 3 7 - 2

into protoporphyrin IX by the enzyme ferrochelatase, to form heme. 8-Aminolevulinic acid dehydrase of erythro- cytes is particularly sensitive to inhibition by lead, since lead interacts with the sulfhydryl groups of this enzyme (Sassa, 1978). In long-term lead exposure, the conversion of protoporphyrin IX into heine by the mitochondrial enzyme ferrochelatase is inhibited. As a result, accumula- tion of &aminolevulinic acid in urine and liver, zinc protoporphyrin IX in erythrocytes, and protoporphyrin IX in liver is observed.

Lead is absorbed primarily through the gut (Mahaffey, 1981) and is found in erythrocytes. Lead moves quickly into and out of soft tissues and accumulates in deep compartments such as bone and teeth, resulting in a very long half-time (months to years) (Marcus, 1985). It is excreted from the body via the kidneys. The effect of lead on cognition and behavior is demonstrated mainly during the developmental stages of the central nervous system

336 O. Fonm et a l . / Eur J. Pharnmcol. Era,iron. Foaicol. Pharmacol Sectton 293 tl995) 335-339

(Goyer, 1993). In rats, it has been clearly shown that lead intoxication alters behavioral stereotypy due to its effects on dopamine levels in various brain regions (Shafiq-ur-Re- hman, 1991).

Benzodiazepines have anxiolytic, anticonvulsant, mus- cle-relaxant, and hypnotic properties. These therapeutic effects are mediated via the central benzodiazepine recep- tors, which are coupled to y-aminobutyric acid receptors and the chloride ion channel (Tallman et al., 1980). Be- sides the central benzodiazepine receptors, there is another type of benzodiazepine receptor, namely, peripheral-type benzodiazepine receptors which are located on peripheral tissues and glial brain cells (Verma and Snyder, 1989). peripheral benzodiazepine receptors are located mainly on the mitochondrial outer membrane (Anholt et al., 1986), although they are also found in erythrocytes, which do not contain mitochondria (Olson et al., 1988). peripheral ben- zodiazepine receptors bind with nanomolar affinity the benzodiazepine ligand Ro 5-4864 as well as the isoquino- line carboxamide derivative PK 11195 (Le Fur et al., 1983). Naturally occurring porphyrins (especially the di- carboxylic porphyrins) such as protoporphyrin IX possess high affinities for peripheral benzodiazepine receptors and have been suggested to be the endogenous ligands for peripheral benzodiazepine receptors (Verma et al., 1987).

Since lead intoxication is associated with elevation of porphyrins and porphyrins are putative endogenous ligands of peripheral benzodiazepine receptors (Verma et al., 1987), we assumed that lead intoxication would affect the expres- sion of peripheral benzodiazepine receptors. Furthermore, we hypothesized that coadministration of PK 11195 might affect the changes in peripheral benzodiazepine receptors induced by lead per se, due to its putative antagonistic activity at peripheral benzodiazepine receptors. The aim of the present study was to investigate the effect of experi- mental acute lead poisoning on peripheral benzodiazepine receptors and the modulatory effect of coadministration of PK I 1195 on the changes induced by lead.

2. Methods and methods

2.1. Materials

[3H]PK 11195 (85 Ci /mM) and [3H]Ro 15-1788 (83.3 Ci /mM) were purchased from New England Nuclear (Boston, MA, USA). Unlabeled PK 11195 was a generous gift from A. Bouvier (Rhfne-Poulenc Santr, Vitry-sur- Seine, France). Unlabeled clonazepam was kindly supplied by H. Gutmann and E. Kyburz, Hoffmann-La Roche (Basel, Switzerland). Lead acetate was purchased from Merck (Darmstadt, Germany). Lumax was obtained from Lumac (Schaesberg, Netherlands). 6-Aminolevulinic acid-HCI was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were purchased from commer- cial sources.

2.2. Treatment schedule

The effects of acute lead poisoning on porphyrin pro- duction and benzodiazepine receptors were studied in adult male Wistar rats (body weight approximately 250 g). The rats were divided into five groups according to the follow- ing treatment schedule: • Group 1 was treated for 3 consecutive days with i.p.

injection of 50 mg/kg solution of lead acetate (Harada et al., 1990) dissolved in dimethyl sulfoxide (DMSO).

• Group 2 received the same treatment as group 1, but lead treatment was discontinued for 7 days.

• Group 3 was treated with a combination of lead acetate and PK II 195 (15 mg/kg, i.p.) for 3 days.

• Control group 1 was treated with PK 11195 alone (15 mg/kg, i.p.) for 3 days (control group IA). Half of this group (control group I B) underwent withdrawal for 7 days.

• Control group 2 was treated with DMSO alone for 3 days (control group 2A). Half of this group (control group 2B) underwent withdrawal for 7 days. After the last injection, each rat was fasted for 24 h in a

metabolic cage, and its urine and stools were collected. The rats were killed by decapitation, and peripheral organs (kidney, adrenals, and liver) as well as cerebral cortex were removed and frozen immediately at -70°(2 until assayed for receptor binding.

2.3. Urinary 6-aminolevulinic acid determination

&Aminolevulinic acid determination was performed as described previously (Tomokuni and Ogata, 1972). Briefly, the &aminolevulinic acid of the urine is reacted with ethyl acetoacetate to produce 2-methyl-3-carbethoxy-4-(3-pro- pionic acid) pyrrole. This product is extracted with ethyl acetate and the aminolevulinic acid-pyrrole absorbance is determined at 553 nm by its reaction with a modified Ehrlich's reagent.

Calculation was performed as follows: (test ab- sorbance/standard absorbance) × 5 = mg/I 6-aminolevu- linic acid. 6-Aminolevulinic acid level was expressed in micromolar concentration according to the following con- version formula: (mg/I × 1000)/131 = m g / l × 7.63 = g,M.

2.4. Determination of lead levels in liver

Liver tissue was wet ashed with concentrated super-pure HNO3 (Merck) (Berman, 1980). The volume of the ashed tissue was brought to 3.3 ml with saline. 2 ml of the concentrate or an appropriate dilution in water of the liver solution were brought to pH 3-4. Lead acetate was chelated with ammonium pyrrolidine dithiocarbamate and extracted into 2 ml of methyl isobutyl ketone. Lead concentration in the methyl isobutyl ketone extract was determined by furnace atomic absorption using Perkin-Elmer 5000 atomic

o. Fonia et aL / Fur. J. Pharmacol. Environ. Toxicol. PharmacoL Section 293 (! 995) 335-339 337

absorption spectrophotometer with Zeeman background correction.

2.5. Receptor binding analysis

Membrane preparation The organs were thawed and homogenized in 50 vol-

umes of 50 mM Tris-HCl buffer, pH 7.4, with a Brinkmann polytron (setting 10) for 10 s at 4°C and centrifuged at 49000 × g for 15 min at 4°C. The pellet was homogenized in the same buffer.

Binding assay [3H]PK 11195 binding was performed as previously

described (Gavish et al., 1987). Briefly, the binding assay contained 400 btl membranes from kidneys (5 mg/ml), adrenals (0.75 mg/ml), or liver (20 mg/ml) and 25 /zl [3H]PK 11195 (final concentration 0.2-6 riM) in the ab- sence (total binding) or presence (non-specific binding) of 1 /zM unlabeled PK 11195 (75 /xl), up to a final volume of 500 /zl. After incubation for 60 min at 4"C, samples were filtered under vacuum over Whatman G F / B filters and washed three times with 3 ml Tris buffer. The filters were placed in vials containing 4 ml of Lumax and counted for radioactivity in a B-scintillation counter after 12 h.

[3H]Ro 15-1788 binding assay was conducted in a final volume of 500 /zi containing 400 p.I of cerebral cortex membranes and 50/xl of [3 H]Ro ! 5- ! 788 (final concentra- tion 0.2-6 riM) in the absence (total binding) or presence (non-specific binding) of 1 pM unlabelled clonazepam (50 btl). The rest of the procedure was as described for [3H]PK 11195 binding. The maximal number of binding sites (Bma ~) and K d values were calculated from saturation curves of [3H]PK 11195 and [3H]RoI5-1788 binding, us- ing Scatchard analysis.

2.6. Statistical analysis

One-way analysis of variance followed by Bonferroni post-hoe test was used for data analysis. Results are ex- pressed as means + S.E.M.

3. Results

3.1. Binding analysis

As shown in Fig. 1A, hepatic peripheral benzodiazepinc receptor density was significantly decreased in group 1 (40% versus control group 1 and 49% versus control group 2) and group 3 (59% versus control group 1 and 51% versus control group 2) (F (4 ,23 )= 8.803, P f0 .0002) . No differences were found between control groups IA and IB or between control groups 2A and 2B. Thus, these four groups were pooled into two groups: control group 1 and control group 2. Lead acetate administration and with-

A 2

"2 1.5

~ 0 5

B 10

o x Q. tq

E

DMS0 Pb PK Pb+PK Pb/W

DMSO Pb PK Pb+PK Pb/W

C 40

~.. 301 c-

="¢~ 20

v lO

DMS0 Pb PK Pb+PK Pb/W

Fig. I. Bin, values of [3H]PK 11195 binding in the liver (A), kidney (B), and adrenal (C) of the various rat groups. [sH]PK 11195 binding was assayed at six concentrations (0.2-6 nM) in the absence (total binding) or presence (non-specific binding) of I p.M unlabeled PK I 1195. The results of each animal were analyzed individually. Results are expressed as meaus+S.E.M. DMSO, control group 2; Pb, group 1; PK, control group I; Fb+PK, group 3; Pb,fW (withdrawal), group 2. " P < 0.05, " " P < 0.01, compared with controls (one-way ANOVA).

drawal did not affect either renal or adrenal peripheral benzodiazepine receptor density: renal, F(4,22)= 2.679, P ffi 0.06; adrenal, F(4,21) = 1.598, P = 0.21 (Fig. IB and Fig. IC). The K d values of kidney, liver, and adrenal were in the nanomolar range (I-4 nM) and did not differ statistically (liver, F(4,23) --- 2.017, P = 0.12; kidney,

F(4,22) = 0.728, P = 0.58; adrenal, F ( 4 , 2 1 ) = 1.273, P = 0.312).

The Br~ x values of [3H]Ro 15-1788 binding to mem- branes from cerebral cortex are shown in Fig. 2. There were no statistically significant differences in the density of central benzodiazepine receptors among the various groups (F(4 ,24) = 0.065, P > 0.8). The K d value of the cerebral cortex was in the nanomolar range ( I - 4 nM) (F(4 ,24) = 0.836, e = 0.52).

3.2. ~ - A m i n o l e v u l i n i c a c i d l eve l s in ,Lrine

"6 E

0 .c

.c_

._I

.q: I

6-Aminolevulinic acid levels in rat urine were deter- mined as an indicator of lead toxicity and are shown in Fig. 3. Statistically significant increases in 6-aminolevu- linic acid levels were observed in groups 1, 2, and 3 (175%, P < 0 . 0 0 1 ; 140%, P < 0 . 0 5 ; and 118%, P < 0.05, respectively, versus control group 2). Only group I showed a significant increase when compared to control group 1 (159%, P < 0.01) ( F ( 4 , 3 l ) = 9.453, P < 0.001).

3.3. L e a d leve ls in l i ver

40

30

20

i0

2 0

;&4t

DMSO Pb

'It'

PK Pb+PK Pb/W

Fig. 3. 6-Aminolevulinic acid levels in urine of the various rat groups. Results are expressed as means 4- S.E.M. For abbreviations, see legend to Fig. I. P<0.05; ' ' P<0.01; " ' ' P<0.001.

P < 0.001; group 3 versus control group 1, 100 + 22 ver- sus 0.15 4-0.06 / z g / g wet weight, P < 0.001). The same was true when group 1 was compared to groups 2 and 3 ( 100 4- 22 versus 40.65 4- 3 . 6 / x g / g wet weight; P < 0.01 ).

Lead levels in rat liver were determined in order to ascertain lead toxicity, groups ! and 2 were combined due to similar results and in order to increase the sample size (n = 6 ) of this group. The same was true for control groups IA and IB (n = 5) and control groups 2A and 2B (n = 8). Lead acetate treatment resulted in a significant increase in lead levels compared to the DMSO and PK 11195 treatments (40.65 + 3.6, 0.115 + 0.03, and 0.15 + 0.06 p , g / g wet weight, respectively; P < 0.05). The com- bined treatment of lead and PK II 195 (n = 5) induced a robust increase in lead levels compared to DMSO (n = 8) and PK ! 1195 (n = 5) treatments (group 2 versus control group !, 100 -I- 22 versus 0.115 4- 0.03 p , g / g wet weight,

O.C

1.5 C

0 x ~ .

l.O

E Q . "-" 0.5

DMSO Pb PK Pb+PK Pb/W

338 o. Fonia et al. / Eur J Pharmacol. Envtron Toxtcol Pharmacol. Section 293 t l995~ 335-339

Fig. 2. Brnax values of [3H]Ro 15-1788 binding in the cerebral cortex of the various rat groups. [3H]Ro 15-1788 binding was assayed at six concentrations (0.2-6 nM) in the absence (total binding) or presence (non-specific binding) of I ,u,M unlabeled clonazepam. The results of each animal were analyzed individually. Results are expressed as means 4- S.E.M. For abbreviations, see legend to Fig. I.

4. Discussion

Lead poisoning is a frequent problem among industrial and arts-and-crafts workers. Absorption of ingested lead from water, food, or dust is the major source of lead in non-occupationally exposed individuals (Lockitch, 1993). The interaction of lead with the heme biosynthetic path- way illustrates how this pathway may be used as an indicator of exposure to toxic agents. As a result of inhibition of 6-aminolevulinic acid dehydrase in erythro- cytes, liver, and kidney, an increase in 6-aminolevulinic acid in the urine of lead-poisoned individuals is observed (Marks, 1985).

In the present study we utilized a simple and rapid model of acute lead poisoning in rats. In this model we obtained within 3 days vastly elevated lead levels in liver and increased 6-aminolevulinic acid excretion in urine, which indicate occurrence of lead poisoning, and a selec- tive decrease of hepatic peripheral benzodiazepine recep- tors. The lead levels in rat livers in the Pb + PK group were significantly higher in comparison to the control groups and to the lead-treated group.

We had hypothesized that PK 11195 administration to lead-poisoned rats would prevent the harmful effects of excessive accumulation of porphyrins in liver induced by lead, since PK 11195 displaces protoporphyrin IX from peripheral benzodiazepine receptors. However, PK 11195 administration to lead-poisoned rats was not protective. Furthermore, it caused over-accumulation of lead in hepa- tocytes, without augmentation of urinary 6-aminolevulinic acid excretion. It seems that PK 11195 amplifies lead accumulation in liver cells, in an unknown mechanism of

O. Fonia et al. / Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Section 293 (1995) 335-339 339

action. It was noted that the increase in urinary &amino- levulinic acid in both the Pb and the Pb + PK groups was parallel to a corresponding decrease in hepatic peripheral benzodiazepine receptors. The decrease in density of pe- ripheral benzodiazepine receptors in liver might be related to the accumulation of &aminolevulinic acid in hepato- cytes in lead-intoxicated rats, which causes damage to liver mitochondria. &aminolevulinic acid autoxidation gener- ates reactive oxygen radicals, which further contributes to liver damage (Hermes-Lima, 1991), mitochondrial injury, and decrease in mitochondrial peripheral benzodiazepine receptors. Morphological studies have demonstrated that lead by itself causes liver damage, due to its accumulation in endothelial and Kupffer cells (Russo et al., 1988).

Unstained samples from lead-treated rats showed small particles (2-5 nm in diameter) of very high electron density which appeared to represent a deposited form of lead ions. These particles are widely distributed in the cytoplasm when morphological injury is apparent (Russo et al., 1988). This injury to liver cells and the accumulation of excess protoporphyrin IX (also caused by lead poison- ing), which is very lipophilic and interacts with mem- branes and proteins (Rademakers et al., 1991), may also have contributed to the decrease in number of peripheral benzodiazepine receptors.

Alternatively, it is possible that protoporphyrin IX is an endogenous ligand for peripheral benzodiazepine recep- tors, and its over-accumulation in liver (which is its major eliminating organ) probably exposed the hepatic peripheral benzodiazepine receptors to its agonistic activity, thus leading to desensitization of these receptors as expressed by the down-regulation.

Acknowledgements

This study was supported by grant 181-103 from the Chief Scientist's Office of the Israel Ministry of Health (to M.G. and R.W.) and grant 181-392 from the Technion V.P.R. Fund-R. Lawrence Research Fund (to M.G.). We thank Miss Ruth Singer for editing the manuscript.

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