Journal ol Tongfi Medical University 9(2):81-85, 1989 8i

The Effects of Cadmium, Mercury and Lead vitro on Hepatic Microsomal Mixed Function Oxidase and Lipid Peroxidation

in

XIAO Gong-hua ( ~ a h ~ ) , WU Jing-long ( ~ ; ~ , ) , LIU Yu-gu (~,]r Department ol Environmental Toxicology, Tongfi Medical University, Wuhan

Summary: In this study, the authors investigated the effect of cadmium, mercury and lead in vit~'o on hepatic mic:osomal mixed function oxidase and lipid per- oxidation. The results showed that these metals could inhibit the activity of hepa- tic mic~'osomal aniline hydroxylase, dee,'ease tho concentration of hepatic mie;oso- real eytochromeP-450 and inc'zease the concentration of the inactive form of hemo- protein, P-420. Besides, they could enhance hepatic mic'~x~somal lipid peroxidation. There were marked coneenti'ation-dependent responsesin these miczosomal reac- tions. Thus, it may be suggested that cadmium, m~ceury and lead are capable of impairing hepatic mic'~'osomal mixed function oxidase in vitro by stimulating membrane lipid peroxidation.

Key words: cadmium, mercury, lead, eytoctu-ome P-450, lipid peroxidation

Lipid peroxida t ion of b i o m e m - branes is a destruct ive phenomenon concerning va r ious cell damages. For example, the microsomal membrane d i s - integration and the dramat ic i n h i b i t i o n of mierosomal mixed func t ion oxidase may be caused by toxic l ipoperoxida t ion products E 1, z 7.

Cadmium, mercury and lead are inhibitors of hepatic microsomal mixed function oxidase. The experimental re - sults showed that these metals decreased microsomal cytochrome P-450 content and the ac t iv i t ies of e thy lmorph ine N - demethylase, a m i n o p y r i n e N-demethylase and anil ine hydroxy la se in the rat E33. Furthermore, i t has been shown that cadmium, mercury and lead could s t i m u - late lipid peroxida t ion of l iver whole homogenate and isolated hepatocytes

t4-~], a l though li t t le was reported on microsomal l ip id peroxida t ion s t imulated by them ~7~. It was reasonable to expect these metals could i nh ib i t drug o x i d a - t ion b y s t imula t ing the peroxida t ion of endoplasmic ret iculum membrane l ip ids . In the present paper we at tempted to inves t iga te the role of l ip id peroxida t ion in the effects of cadmium, mercury and lead in v i t r o on microsomal mixed funct ion oxidase.

METHODS

1. I so la t ion of microsomes 20 male wi s t a r rats, weighing 180-220

g, were killed by decapi tat ion, and the I ivers were qu ick ly removed and washed several t imes in ice-cold 0 . 8 5 ~ sal ine and homogenized in 3 volumes

8~

of 150 m KCI-10 retool/1 Tris-HCl, pH 7.40, using Potter-Elvehjem homogen- izer. The homogenates were centrifuged at 9 000 g for 20min.Microsomes, obtained from the resulting supernatant fractions by ultracentrifugation at 105 000 g for 1 h, were resuspended in the same buffer. 2 . Enzymatic analysis

Cytoehrome P-450 content and aniline hydroxylase act ivi ty were determined to observe direct effects of cadmium, mer- cury and lead in vi tro on hepatic micro- somal mixed function oxidase. Aliquots of the microsomal suspension were pre- incubated with cadmium, mercury or of lead (10-7-10-2mol/1) at 37"C and 80 oscillations/rain in a metabolic shaker of 20 min. Cytochrome P-450 and cytochro- me P-420 werc quantitated by the Omu- ra and Sato method ta~. Aniline hydro- xylase act iv i ty was determined by the method of O 1 Brien et al cgJ. 3. Peroxidation of microsomes

Cadmium acetate at final concentra- tions of 500- 10 000 ~mol/l mercuric chlo- ride at final concentrations of 10-100 pm- ol/1 and lead acetate at f inal concentrations of 25-1 000 pmol/1 were added to the incu- bation flasks containing incubation mixture, at zero time. The incubation mixture consisted of 1.0 ml hepatic microsomal suspension (about 1 mg protein), and 4.0 m1150 retool/1 KCI-10 retool/1 Tr i s -HCi pH 7.40. Incubations were carried out at 37~ and 80 oscilla- t ions/ra in in a metabolic shaker. Al i - quots of the microsomal suspension were removed at 10, 20, 30, 45, 60 and 75 min for assay of lipid peroxidation. Lipid peroxidation was quantitated by spectrophotometric determinations of malonaldehyde-thiobarbituric acid (TBA) The assay was based on the method of Staccy~ 1 o 4. Protein determination and sta- tistics

Protein was determined by the me- thod of Lowry e~ al ~117. Statistical analysis was made by a one tailed t test. Each data point represents a mean

value obtained from 3-7 experimentS.

RESULTS

1. Effects of cadmium, mercury and lead in vi tro on microsomal mixed func- t ion oxidase

1) Aniline hydroxylase act ivi ty The act ivi ty of aniline hydroxylase was

decreased by cadmium, mercury and lead, depending upon their concentra- tions in the mixtures. When microsomes were preincubated with cadmium (10-4 to 10-2 mol/1), mercury (10 -~ to t 0 -s mol/1) or lead (10 -4 to 10 -~ mol/1), aniline hydroxylase act iv i ty was significantly inhibited. The results are presented in f ig .1 . It was found that the metals were capable of directly inhibit ing he- patic microsomal aniline hydroxylase

Fig. 1. Effects of varying in vitro concentra- tions of the metals on hepatic micro- somal aniline hydroxylasc activities. The control value of aniline hydro- xylasc without addition of metals is 18.1-21.4 P-aminophenol/mg protein / I min.

act ivi ty . Estimated ICsQ values ( i . e . the concentrations of these metals re- quired to inhibi t control by 50 4o) ranged from 340 to 540 ~mol/1 (cadmium), 10 to

Journal o] Tongjf Medical University 9(2):81-85 1059 83

28 ~mol/1 (mercury) , and 450 to 770 pmol/1 (lead) respectively.

2) Cy tochromeP-450 content Since the addit ion of cadmium, mercury or lead to liver microsomes produced a concentration-related inh ib i t ion of sub- strate metabolism, it was of interest to determine whether the i nh ib i to ry ef- fects of the metals were mediated by con- verting active cytochrome P-450 into the inactive form of the hemoprotein, P-420. The results of the experiment are illustrated in fig. 2. Addi t ion of Cd §247 Hg § or Pb §247 to liver mierosomes in increasing concentrat ions caused a concentration-dependent loss of detect- able cytochrome P-450 that was accom-

panied by increasing format ion of P - 420. These apparent conversions of cytochrome P-450 into P-420 were markedly enhanced in the presence of Cd +§ or Pb +§ concentrat ions greater than 5 • 10- 5 tool/1 (50 ~mol/1) and Hg §247 concentrat ions greater than 10- ~ mol / l (10 ~tmol/1). The levels of eytochrome P-450 detected in microsomes treated wi th • tool/1 (500 ~tmol/1) Cd §247 5 • 10-5mol/1 (50 ~tmol/1) Hg +§ or 10 -3 tool/1 (1000 pmol/1) Pb ++ were about 50 ~ of those ob- served in untreated microsomes. In add i - t ion, the conversion of cytochrome P-450 into P-420 by Cd ++ or Hg +§ was not stoichometric, since a loss of total spectrophotometrically detectable c y t o -

='g ~ o

o .~ O. 50F ~ - /

o ~ / . _ a - e " ~ - - .

~ � 9 / A

0" 10 .6 10 - s 10 -4 I0 - s 10-"

Cd++(M)

I_ 6 10 I0 ks 10 J-4 IOLS

Pb++(M)

Fig.2. Effects of varying in vitro concentration of cadmium, mercuzy and lead on microsomal cytoehrome P-450 and eytoehrome P-420 cont ents. C)=tcytoehrome P-450 /k eytochtome P-420

cytochrome P-450+P-420;

hepatic

Cd++(uM) pb++luM) A ~ l O ~ J O

"~ ~ looo "~ 25

.~ 500

~ Control ol . ol

~ ' ' 0 ~ ~ 3 ~ 4 ~ 6b 7 ~ - Incubation time (min)

Fig.3. Stimulation of mierosomal lipid peroxidation by varying in vitro concentrations of cadmium, mercury and lead.

84

chrome accompanied the enhanced act ivi ty of cytochrome P-450.

2. Effects of cadmium, mercury and lead in vi tro on hepatic microsomal lipid peroxidation

Cadmium-, mercury- and lead- induced lipid peroxidation, as assessed by the determination of the malonalde- hyde--TBA complex, is shown in fig.3. A concentration- and time-dependent increase can be noted. The levels of lipid peroxidation detected in microsomes pre- incubated with 500 to 1000 l~mol/1 Cd §247 10 to 25 ~mol/1 Hg +§ or 50 to 100 l~mol/1 Pb §247 were about twice as high as those observed in untreated microsomes.

DISCUSSION

1. Effects of cadmium-, mercury- and lead-stimulated lipid peroxidation on microsomal mixed function oxidase

A number of investigators have demonstrated that in vitro microsomal lipid peroxidation severely decreases cytochrome P-450 content and mixed function oxidase activityL1,1~] For example, Hruszkewycz et al demonstrat- ed that lipids extracted from peroxidized microsomes destroyed 61% of the cyto- chrome P-450 content and 73 % of the glucose-6-phosphatase act ivi ty of fresh mierosomes EI~. Strong correlations be- tween the extent of peroxidation and the decline in aminopyrine demethylase or aniline hydroxylase activities have been observed for NADPH-dependent, ascor- bate-promoted, ferrous iron-stimulated and carbon tetrachloridc-induced lipid peroxidation E 12

In the present series of experiments, pcadmium, mercury and lead produced a dose- and time-dependent increase of lipid peroxidation. There were strong correlations between the extent of per- oxidation and the decline in cytochrome P-450 content and aniline hydroxylase act ivi ty. For example, when liver micro- somes from untreated rats were incu- bated with 10 -3 mol/1 (1000 ~tmol/l) Cd +§ the peroxidation of hepatic rnicrosomal

membrane lipids was elevated by as many as 3.8 times and microsomal P - 450 content and aniline hydroxylase act ivi ty were decreased by as much as 5 5 ~ and ~3~ respectively after 20 rain of incur a t ~ on; the presence of 10- 4 mol/1, (100 ~tmol/1) Hg § in the incubation mixture resulted in an increase of micro- somal lipid peroxidation by as many as 3.2 times and a decrease of microsomal cytochrome P-450 content and aniline hydroxylase activities by as much as 5 9 ~ and 64~ respectively after 20 min of incubation; and the presence of 10 -3 tool/1 (1000 pmol/1) Pb +§ in the incubation mixture resulted, in an in - crease of microsomal lipid peroxidation by as many as 1.6 times and a decrea- se of microsomal cytochrome P-450 con- tent and aniline hydroxylase activities by as much as 48~ and 5 5 ~ respec- tively (shown in fig. 1-3) . If only the extent of lipid peroxidation is consider- ed without taking into account the kinds and concentrations of the metals which have been added to the incubation mixtures, it may be seen that the higher the extent of lipid peroxidation is stimulated, the more depressing the con- tent of cytochrome P-450 and act ivi ty of aniline hydroxylase in microsome are. From the above it may be suggest- ed that stimulating microsomal lipid peroxidation may be one of the mechan- isms by which cadmium, mercury and lead inhibit hepatic microsomal mixed function oxidase directly.

2. Effects of cadmium-, mercury- and lead-stimulated lipid peroxidation on conversion of cytochrome P-450 into P-420

The addition of cadmium, mercury and lead to liver microsomes produced a concentration-related inhibit ion of an i - line metabolism and increase of convert- ing active cytochrome P-450 into the inactive form of hemoprotein, P-420. The inhibi t ion of mixed function ox id- ase which accompanies microsomal l i - pid peroxidation may be caused in two ways, by damage of membrane structural

Journal ol Tongji Medical University 9(2):81-85, 1989 85

integri ty and direct des t ruc t ion of c y t o - chrome P-450 heine, and by tox ic l i p o - ~ r o x i d a t i o n products . In suppor t of cytoehrome P-450 des t ruc t ion , H r y c a y and O' Brien rx3: demonstra ted that l i n o - leie acid hydrope rox idase converts c y t o - chrome P-450 in to cytochrome P-420 during pe rox idase -ca t a lyzed decompo- s i t ion of the hydroperox ide . Moreover, Jeffery et a l ~4~ showed that the a d d i - t ion of exogenous l ino le ic acid h y d r o - peroxide to control mierosomes in vi tro resulted in a concen t r a t i on -depen - dent des t ruc t ion of cytochrome P-450 and a p a r a l l e l l o s s of mixed func t ion oxidase ac t i v i t i e s which w e r e a c c o m p a n - ied by an increase of cy tochrome P - 420. However , the ques t ion whether c ad - mium- , m e r c u r y - and l ead - s t imula t ed in v i t ro hepat ic mic rosomal l i p id pe r - oxida t ion w i l l b r ing about an inerease of the content of l inole ic acid h y d r o - peroxide remains to be fur ther i n v e s t i - gated.

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