Mechanism of chloroform nephrotoxicity

TOXICOLOGY

AND APPLIED

73, 5 1 l-524 (1984)

PHARMACOLOGY

Mechanism of Chloroform III. Renal

Department

and Hepatic

Microsomal

Nephrotoxicity

Metabolism

of Chloroform

JACQUELINE

H. SMITHY AND JERRY B. HOOKY

of Pharmacology Michigan State

and Toxicology, University, East

Received

August

19, 1983;

Center for Environmental Lansing, Michigan 48824

accepted

December

in Mice’

Toxicology,

20, 1983

Mechanism of Chloroform Nephrotoxicity. III. Renal and Hepatic Microsomal Metabolism of Chloroform in Mice. SMITH, J. H., AND HOOK, J. B. (1984). Toxicol. Appl. Pharmacol. 73, 5 1 l-524. In vitro studies with male ICR mouse renal cortical slices have indicated that chloroform (CHCl,) is metabolized by the kidney to a nephrotoxic intermediate, possibly by a cytochrome P45Odependent mechanism similar to that occurring in the liver. In this investigation, metabolism of %HCls by microsomes prepared from renal cortex and liver provided definitive evidence for a role of cytochrome P-450 in the renal metabolism and toxicity of CHCls . i4CHClp was metabolized to ‘%O, and covalently bound radioactivity by male renal cortical microsomes; metabolism required oxygen, a NADPH regenerating system, was dependent on incubation time, microsomal protein concentration, and substrate concentration, and was inhibited by carbon monoxide. Consistent with the absence of CHCls nephrotoxicity in female mice, little or no metabolism of “CHCl, by female renal cortical microsomes was detected. CHCls produced a type I binding spectrum with oxidized male renal cortical and hepatic microsomes. Incubation of glutathione with microsomes and ‘%HC& increased the amount of aqueous soluble metabolites detected with a concomitant decrease of metabolism to “‘CO2 and covalently bound radioactivity, suggesting the formation of a phosgene conjugate as has been described for hepatic CHCls metabolism. These data support the hypothesis that renal cytochrome P-450 metabolizes CHCl, to a nephrotoxic intermediate.

Chloroform has been investigated extensively as a model hepatotoxicant. Hepatotoxicity requires the metabolism of CHC4 by a cytochrome P-450oxygen-dependent mechanism to phosgene, the presumed reactive intermediate (Mansuy et al., 1977; Pohl et al., 1977,

1980; Pohl, 1979). The extensive information on CHC13-induced hepatic and renal toxicity and the relatively uncomplicated route of metabolism in the liver offers an ideal model toxicant to evaluate biochemical mechanism(s) of nephrotoxicity. Investigations by Paul and Rubinstein ( 1963) indicated that rat kidney slices metabolized 14CHC13 to *4C02 at a rate approximately 5 to 10 times less than liver slices. However, the mouse has provided a more suitable species for mechanistic evaluation of CHC4 nephrotoxicity due to the dramatic sex and strain differences in susceptibility to CHC13 nephrotoxicity (Eschenbrenner, 1944; Culliford and Hewitt, 1957; Hill et al., 1975). Covalent binding of i4CHC13 to male C57Bl/ 65 mouse kidney protein in viva was similar

’ Presented in part at the American Society for Pharmacology and Experimental Therapeutics, Philadelphia, Pa., August, 1983. * Present address: Laboratory of Experimental Therapeutics and Metabolism, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20205. 3 To whom requests for reprints should be addressed: Dr. Jerry B. Hook L-60, Smith Kline & French Laboratories, 1500 Spring Garden Street, Philadelphia, Pa. 19101. 511

0041-008X/84

$3.00

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form ress-wd.

512

SMITH

AND HOOK

to liver binding (Ilett et al., 1973). In contrast, covalent binding of i4CHC13 in vitro to microsomal protein from male mouse liver was approximately 10 times greater than to kidney microsomal protein (Ilett et al., 1973; Clemens et al., 1979). Thus, the relationship between renal metabolism and nephrotoxicity of CHCls has been less apparent. Recent experiments have suggested that CHC& metabolism in the kidney may occur by a pathway similar to that demonstrated in the liver. CHC& nephrotoxicity could be detected 2 hr following CHCL administration to male ICR mice as decreased ability of renal cortical slices to accumulate organic ions and decreased concentrations of nonprotein sullhydryls (an indication of reduced glutathione) (Smith et al., 1983). Nephrotoxicity was not detected in female mice. An in vitro model with renal cortical slices from adult male and female ICR mice indicated that renal toxicity and metabolism of CHCls were related (Smith and Hook, 1983a,b). In this model, CDC13 was less nephrotoxic than CHC13 (Smith and Hook, 1983b). Furthermore, incubation of male mouse renal cortical slices under an atmosphere of 80% carbon monoxide:20% oxygen reduced the in vitro metabolism and toxicity of CHC& (Smith and Hook, 1983a,b). Thus, these experiments suggested that CHCls may be metabolized in the kidney by a cytochrome P-450-dependent mechanism similar to that in the liver. The purpose of this investigation was to assesscomparatively renal cortical and hepatic microsomal metabolism of CHCls in microsomes prepared from male and female ICR mice to test the hypothesis that CHC13 is metabolized by a cytochrome P-450-mechanism in the kidney. METHODS Animals Adult male and female ICR mice (30 g, Harlan Farms, Haslett, Mich.) were maintained on a 12-m light-dark cycle (OSOO-to 2OOQ-hr light) in a temperature and hu-

midity controlled room. Animals were allowed free access to feed (Wayne Lab Blox, Chicago, Ill.) and water. Preparation of Subcellular Fractions and Spectral Measurements Kidneys and livers from mice were quickly excised, pooled, and placed in ice-cold 0.1 M sodium phosphate buffer, pH 7.4, containing 1.15% KCl. After weighing, the medulla and papilla were dissected from the kidneys and discarded. Renal cortex from male and female mice and male livers were minced in this same buffer, rinsed three times, and homogenized in 3 vol of buffer by a Potter-Elvehjem homogenizer with a Teflon pestle followed by centrifugation at 9000g for 30 min. The resulting supematant fraction was then centrifuged at 105,OOOgfor 60 min. The 105,OCOgsupematant fraction was the cytosol fraction. The pellet was resuspended in the same buffer and recentrifirged at 105,000g for 60 min. The microsomal fraction (pellet) was resuspended in 0. I M sodium phosphate buffer, pH 7.4, to a final concentration of IO to 15 mg protein/ml. Microsomal protein concentration was determined by the method of Lowry et al. (195 1). Cytochrome P-450 concentrations were determined from the dithionite-reduced CO difference spectra as described by Omura and Sato (1964). Substrate binding studies were conducted as described by Schenkman et al. ( 1967). Spectral measurements were made on a Beckman dual beam spectrophotometer (Model UV5260). Assessment of ‘“CHC13 Metabolism in Vitro Preparation of’*CHCI,. ‘THCl, (98% purity, Pathfmder Laboratories, St. Louis, MO.) was diluted to a specific activity of approximately 0.5 &i/fimol with nonradioactive CHCl, (99+%, stabilized with 0.75% ethanol, Aldrich Chemical Co., Milwaukee, W&c.). This ‘THCls was diluted with 9 vol of dimethylformamide (Mallinckrodt Chemical Co., St. Louis, MO.). Incubation procedures. Metabolism of 14CHCls (diluted to a specific activity of 0.5 aCi/pmol) by mouse tissue was evaluated by measuring the conversion of “CHCla to r4C02, radioactivity irreversibly associated with the protein fraction (an indication of covalent binding), and aqueous soluble radioactivity. The incubations were conducted in 25-ml Erlenmeyer 8ask.scontaining microsomes and a NADPH regenerating system in a phosphate-bufhered medium, pH 7.4. Specific alterations in the composition of the incubation medium are noted in the figure legends. The flasks were oxygenated with 100% Or for 5 min, “‘CHQ was added directly to the incubation medium, the flaskswere sealed immediately with a sleeve-type rubber septum and were incubated at 37°C in a metabolic shaker (100 cycles/min). Each flask had a removable center well attached to the rubber septum containing a l-cm’ piece

RENAL

CHLOROFORM

METABOLISM

513

of lilter paper with 50 pm01 of NaOH as a CO, trap. The Determination of aqueous soluble ‘*CHC& metabolites. incubation was terminated by the injection of 10% triIn some experiments, the aqueous supematant fraction chloroacetic acid (TCA) in a volume equal to the reaction of the reaction homogenate was extracted with 4 ml diethyl volume with a syringe through the rubber septum. The ether until radioactivity could no longer be extracted from flasks remained sealed for at least 3 hr at room temperature the aqueous phase to remove unmetabolized “CHClI. A after incubation was terminated to allow for trapping of 0.5-ml aliquot of the aqueous phase was counted in 10 ‘%02 as sodium carbonate. In some experiments, the ml of ACS for 10 min, each; quench was corrected by flasks were refrigerated overnight. The rubber scpta were internal standardization with [‘%Y]toluene. Data are exremoved, and the amounts of “COz, covalently bound pressed as nanomoles “CHCI, converted to aqueous solradioactivity, and aqueous soluble radioactivity were uble metabolites per reaction vessel. measured as described below. Nonenzymatic metabolism of “CHClI was assessedin reaction flasks containing heat-denatured microsomal RESULTS protein (lOO”C, 5 min) and, where indicated, was subtracted to obtain corrected values for enzymatic ‘%HClp metabolism. The source of heat-denatured protein was Metabolism of ‘“CHC13 in Vitro by Renal Cortical and Hepatic Microsomes-Eflect of varied between either male liver or male or female kidney; the degree of nonenzymatic metabolism was not influenced Time, Microsomal Protein Concentration, by the source of protein (data not shown). Data are exand Substrate Concentration pressed as i -t SE of the mean. Determination of “CO, production. 14C02 evolution 14CHC13 was metabolized to 14C02 and cowas detected by the method of Pohl et al. (1980). The valently bound metabolites by microsomes filter paper CO2 traps were transferred to 1S-ml screw cap prepared from male mouse kidney cortex and tubes containing 1 ml HzO. Unmetabolized “CHClr was removed by extracting the resulting alkaline solutions with liver (Figs. 1 and 2). Metabolism of 14CHC13 2 ml of diethyl ether until radioactivity could no longer by microsomes prepared from female kidney be extracted for three consecutive washes. This procedure cortex was similar to nonenzymatic “CHC13 usually required seven extractions. A 0.5-ml aliquot of decomposition in heat-denatured (boiled) mithe washed alkaline solution was counted in 20 ml ACS counting scintillant (Amersham Corp., Arlington Heights, crosomes (data not shown). Even when inIll.) for 10 min each; quench was corrected by internal cubation time was extended up to 2 hr, there standardization with [ 14Cltoluene (New England Nuclear, was little or no metabolism of r4CHC13 to Boston, Mass.). Data are expressed as nanomoles 14COz *4C0, or covalently bound radioactivity by detected per reaction vessel per incubation time. female renal cortical microsomes (Fig. 1). Determination of covalently bound radioactivity to proteins. The trichloroacetic acid precipitated protein was “CHClJ metabolism by male renal cortical transferred from the Erlenmeyer flask to a 1%ml screw- microsomes to 14C02 was linear with time for cap tube. The denatured protein was separated by ccn- approximately 15 mitt; metabolism to covatrifugation. The aqueous layer was removed and, in some lently bound radioactivity was linear with time experiments, extracted for determination of aqueous solfrom 5 to 30 min (Fig. 1). Hepatic microsomal uble radioactivity (see below). Radioactivity not covalently bound to proteins was removed as described by Ilett et metabolism of 14CHC13 to 14C02 was linear al. (1973). The protein pellets were washed with 5 ml of with time for 15 min, and metabolism to cohot (50°C) methanokether (3: I) until radioactivity could valently bound radioactivity was linear for 30 no longer be extracted from the pellets for three consecutive min (Fig. 2). r4CHCIJ metabolism to 14C02 washes; this procedure usually required seven extractions. and covalently bound radioactivity always was The protein pellets were dissolved in 1 ml of 1 N NaOH, greater by hepatic than by renal cortical miand an aliquot was taken for determination of protein crosomes from male mice (Figs. 1 and 2). Fur(Lowry et af., 195 1). The covalently bound radioactivity was determined by counting a 0.5~ml aliquot of the alkaline thermore, there was an increase in the magsolution in 10 ml of ACS containing 0.5 ml of I N HCl. nitude of difference between hepatic and renal Samples were counted for 10 min each; quench was corcortical metabolism as incubation time inrected by internal standardization with [‘%Icreased. toluene. Data are expressed as nanomoles i4CHClp coMale renal cortical microsomal metabolism vslently bound per milligram microsomal protein, or were extrapolated to nanomoles “CHClr covalently bound per of 14CHC13 to 14C02 and covalently bound reaction vessel where indicated. radioactivity was linear with protein concen-

514

SMITH

AND HOOK 2.5 r

6 0 ‘i z

2.0

P = e c P “, a E 0 0 6 ”

1.5

1.0

0.5

r + m= 0

n

30

60

Incubation

90

l

0 I 0

120

Time (min)

I 30

1 60

Incubation

I 90

I 120

Time (min)

FIG. 1. Time course of ‘%ZHCIJ metabolism to “‘CO, and covalently bound radioactivity by mate and female renal cortical microsomes. Reaction vesselscontained 2 mg microsomal protein, 3.12 nmol ‘%IHC& (specific activity 0.5 $Zi/pmol, added in a volume of 2.5 pl of dimethylfonnamide), 0.4 pmol NADPH, 1.2 wmol NADP+, 1.2 nmol NADH, 18 nmol gh~cose6-phosphate, 4 units ghxose-6-phosphate dehydrogenase, and 0.65 mmol MgCl, in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 1.5 ml. Values have been corrected for nonenzymatic metabolism of “CHClr with boiled microsomes. Values are X + SE, n = 4. 12.5

- 10.0 .-c 2 e P

a &I u? i! 50 .-s ; i

0 ?!

40

E

30

:

20

0 d

IO

7.5

8 c F i c 5.0 z E 2 ! s 2.5

c {

2

0

30 Incubation

60

90

Time (min)

120

0

30 Incubation

60 Time

90

120

(min)

FIG. 2. Time course of “CHCIj metabolism to W02 and covalently bound radioactivity by male hepatic microsomes. Incubation conditions and data presentation are described in Fig. 1.

RENAL

1 0

CHLOROFORM

I 4

I 2

Microromal

I 6

Protein

515

METABOLISM

I 0

1 0

(mg )

I

’ 2

4

Microsomal

,

6

Protein

0 (mg )

Ftc. 3. Effect of male renal cortical microsomal protein concentration on ‘%HClr metabolism to ‘%O, and covalently bound radioactivity. Reaction vesselscontained 0.5 to 8 mg microsomal protein, 3.12 pmol ‘%ZHCl, (specific activity 0.5 &i/pmol, added in a volume of 2.5 ~1 of dimethylformamide), 0.4 pm01 NADPH, 1.2 pmol NADP+, 1.2 pmol NADH, 18 pmol glucose 6-phosphate, 4 units glucose-6-phosphate dehydrogenase, and 0.65 mmol MgC& in 0.1 M sodium phosphate buKer, pH 7.4, in a total volume of 1.5 ml. Values have been corrected for nonenzymatic metabolism of ‘%HCl, and are extrapolated to nanomoles of metabolite formed per reaction vessel. Values are X f SE; n = 3 to 5 for each data point. to at least 2 mg of microsomal protein during a lo-min incubation with 3.12 pmol 14CHClX (Fig. 3). Metabolism of 14CHC13 to 14C02 and covalently bound radioactivity by hepatic microsomes appeared linear up to 8 mg of microsomal protein, the highest concentration used in these studies (Fig. 4).

tration

On the basis of these data, an incubation period of 10 min with a microsomal protein concentration of 2 mg per 1.5 ml reaction was used for subsequent experiments. Cytochrome P-450 content in microsomes per milligram microsomal protein was approximately four times greater in liver than

- r 40

rv ,o t

OL I

1

I

I

0

2

4

6

Microsomal

Protein

1

0 (mg 1

0

2

Microromal

4 Protein

b

8 (mg)

FIG. 4. Effect of male hepatic microsomal protein concentration on “CHClX metabolism to 14C02 and covalently bound radioactivity. Incubation conditions and data presentation are described in Fig. 3.

516

SMITH

AND HOOK TABLE I

COMPARISON

OF RENAL CORTICAL AND HEPATIC IN RELATION TO CYT~CHROME

MICRO~~MAL

METABOLISM

OF ?THC13

P-450CONCENTRATIONS’

Male

kidney

cortex

Liver

P-450

Cytochrome

nmol/mg protein

0.453 + 0.029

1.621 C!I0.065

Covalent binding nmol/mg protein nmol/nmol cytochrome P-450

1.086 + 0.167 2.435 + 0.410

2.092 f 0.264 1.237 f 0.114

“‘CO2 detected nmol/mg protein nmol/nmol cytochrome P-450

3.468 + 0.360 7.803 k 1.024

6.681 f 0.476 4.103 + 0.245

,JReaction vesselscontained 2 mg microsomal protein, 3.12 pmol ‘%HCl, (specific activity 0.5 &i/pmol, added in a volume of 2.5 pl of dimethylformamide), 0.4 pmol NADPH, 1.2 pmol NADP+, 1.2 pmol NADH, 18 pmol glucose 6-phosphate, 4 units glucose-6-phosphate dehydrogenase, and 0.65 mmol MgCl, in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 1.5 ml. Values have been corrected for nonenzymatic metabolism of r4CHC13 and are expressed as nmol %HCl metabolized/mg microsomal protein or/nmol cytochrome P-450. Values are + SEM, n = 5.

in male kidney cortex (Table 1). Microsomal metabolism of 14CHC13 to covalently bound radioactivity and 14C02 was approximately

two times greater by liver than by kidney after a lo-min incubation of 3.12 pmol 14CHClJ with 2 mg microsomal protein. Therefore, when metabolism was expressed in terms of nanomoles of metabolites per nanomoles cytochrome P-450, CHC& metabolism was approximately two times greater in male mouse

0.6

> 0.4 0

2

4

CHCI2

6

8

IO

12

14

(pmol)

FIG. 5. Effect of substrate concentration on ‘%HCls metabolism to ‘%Oa by male renal cortical microsomes. Reaction vesselscontained 2 mg microsomal protein, 1.25 pmol “CHCls (specific activity 0.5 aCi/pmol) plus 0.25 to 1I .25 pmol nonradioactive CHCls to provide the indicated substrate concentration, 0.4 pmol NADPH, 1.2 pmol NADPC, 1.2 pmol NADH, 18 rmol glucose 6-phosphate, 4 units glucosed-phosphate dehydrogenase, and 0.65 mmol MgCl, in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 1.5 ml. Values have been corm&d for nonenzymatic metabolism of “CHCIB with boiled microsomes. Values are X 1- SE, n = 3.

FIG. 6. Kinetics of the renal cortical microsomal metabolism of “CHQ to ‘m. Substrate concentrations ranged from 1.25 to 12.5 pmol. See Fig. 5 for a description of the incubation condition. Data were plotted by the method of Eadie and Hofstee (&die, 1952; Hofktee, 1952). V = nmol ‘*CO2 detected/reaction vessel/min and S = pmol CHCI, added to the incubation; n = 3; I’,,,, = 0.782 nmol/2 mg microsomal protein/min; K,,,= 2.78 pmol.

RENAL 5

CHLOROFORM

COVALENT BINDING ( nmol I mg protein)

5

4

4

3

3

2

2

1

1

0

517

METABOLISM

(nmol

I reaction

vessel )

0 Boiled -New” NADH NADPH NAM+ -NADP” NADPH

Bdd

-NADH NADH NADPH NADHI -NADPH NADPH

FIG. 7. Effect of NADH and NADPH on male renal cortical metabolism of ‘%HCls to covalently bound radioactivity and ‘%02. All reaction vessels contained 1 mg microsomal protein and 3.12 pmol “CHCl~ (specific activity 0.5 pCi/amol, added in a volume of 2.5 ~1 of dimethylformamide) in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 0.75 ml (-NADH - NADPH). Reaction vessels also contained 1 mM NADH (NADH), 1 mM NADPH plus 1 mM NADP+ (NADPH), 1 mM NADH plus 1 mM NADPH plus 1 mM NADP+ (NADH + NADPH) as indicated, plus 3 mM MgCl,, 5 mM glucose 6phosphate, and 1.5 units glucosed-phosphate dehydrogenase. Boiled reaction vesselswere similar to NADH + NADPH flasks, but were boiled for 5 min. Values are X f SE; n = 4.

renal cortical microsomes than in hepatic microsomes (Table 1). Male mouse renal cortical metabolism of 14CHC13 to 14C02 increased linearly with increasing substrate concentration up to 5.0 pmol of CHC& (Fig. 5). An Eadie-Hofstee plot (Eadie, 1952; Hofstee, 1952) was constructed to estimate the kinetic parameters for the renal cortical microsomal metabolism of CHC13 (Fig. 6). The Michaelis constant (K,) for the reaction was 2.78 pmol and V,,,, was 0.782 nmol/2 mg microsomal protein/min. COVALENT BINDING (nmol I mg protein)

On the basis of these data, the substrate concentration was increased in subsequent reactions to decrease the potential that CHCl3 concentrations would become rate-limiting. Effect of NADH and NADPH on Microsomal Metabolism of 14CHC13 Metabolism of “CHC13 by renal cortical and hepatic microsomes required the presence of a NADPH regenerating system (Figs. 7 and ‘to, (nmol

DETECTED I reaction

vessel

1

FIG. 8. Effect of NADH and NADPH on male hepatic microsomal metabolism of ‘%HCls to covalently bound radioactivity and i4COz. Incubation conditions and data presentation are described in Fig. 7.

518

SMITH AND HOOK

8). The extent of 14CHC13metabolism to 14C02 and covalently bound radioactivity was similar in incubations with heat-denatured microsomes or with microsomes in the absence of a NADPH regenerating system. Enzymatic metabolism of 14CHC13 by renal cortical microsomes in the presence of NADH alone was only about 5% of that observed in the presence of NADPH (Fig. 7). In contrast, hepatic microsomal “CHC13 metabolism in the presence of NADH alone was approximately 40% of that observed in the presence of NADPH alone (Fig. 8). Addition of both NADH and NADPH to the incubation mixture increased the metabolism of “CHC13 in an additive manner.

Subcellular Locations of ‘“CHC13 Metabolism Hepatic and renal cortical 90008 supernatant fraction and the microsomal fraction from male ICR mice metabolized 14CHC13 to 14C02 and covalently bound radioactivity (Table 2). Metabolism of 14CHC13 by the cytosol fraction was very low. There was little or no conversion to aqueous soluble metabolites by the microsomal fraction alone; however, when glutathione was added, the amount of aqueous soluble counts was increased. There was no metabolism of 14CHC13 by any of these fractions in the absence of a NADPH regenerating system (data not shown).

TABLE 2 METABOLISM OF %HCl, TO 14C02, COVALENTLY IN THE PRESENCE AND ABSENCE OF GLUTATHIONE FRACTIONS ’

BOUND RADIOACTIVITY, (GSH) BY MALE RENAL

AND AQUEOUS SOLUBLE CORTICAL AND HEPATIC

Renal cortex -GSH

METAB~LITES SUBCELLULAR

Liver +GSH

-GSH

+GSH

1.226 f 0.385

2.895 + 0.422 2.059 f 0.141 0.503 f 0.135

2.446 + 0.307 0.747 + 0.091 2.186 f 0.316

9000 g supematant fraction 14COzb Covalent binding’ Aqueous’

1.487 f 0.304 0.518 f 0.169 0.092 + 0.041

Cytosol 14C02 Covalent binding Aqueous

0.009 f 0.005 0.016 + 0.004 0.012 f 0.012

0.110

0.006 f 0.006 0.053 f 0.043

0.252 f 0.226 0.098 k 0.066 0.011 + 0.008

0.132 f 0.052 0.027 + 0.018 0.212 + 0.061

Microsomes ‘co, Covalent binding Aqueous

1.672 + 0.344 0.365 f 0.109 0.012 f 0.008

0.939 f 0.225 0.116 f 0.025 1.582 f 0.470

4.104 + 0.477 1.929 + 0.334 0.014 + 0.014

3.715 + 0.654 0.379 + 0.104 6.287 + 1.000

Microsomes + cytosol 14C02 Covalent binding Aqueous

1.651 f 0.458 0.396 + 0.165 0.074 f 0.024

1.175 + 0.358

0.222 -t 0.054

5.419 + 0.333 3.054 + 0.463 0.392 + 0.123

4.376 + 0.342 0.790 * 0.07 1 5.512 -t 0.878

0.198

f 0.057

0.778 f 0.239 f 0.110

1.112 + 0.392

@Reaction vessels contained 2-mg 9000g supematant fraction, or 1 mg cytosolic, or 1 mg microsomal, or 1 mg cytosolic plus 1 mg microsomal protein, and 5 mM glutathione (GSH) as indicated. In addition, reaction vessels contained 3. I2 amol ‘%HC& (specific activity 0.5 pCi/amol, added in a volume of 2.5 d of dimethylformamide), 0.1 pmol NADPH, 0.3 pmol NADF, 0.3 pmol NADH, 4.5 pmol glucose 6-phosphate, 1 unit glucose&phosphate dehydrogenase., and 0.16 mmol MgCl, in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 0.75 ml. Values have been corrected for nonenzymatic metabolism of i4CHCla by incubations without a NADPH regenerating system. Values are X * SE, n = 4. b nmol detected/reaction vessel. c nmol/reaction vessel.

RENAL

CHLOROFORM

r

+0.02

Binding Spectra of CHCI, with Renal and Hepatic Mcrosomes The binding of CHCIJ to oxidized cytochrome P-450 from male renal cortical and hepatic microsomes produced a typical type I binding spectrum (Fig. 9).

519

METABOLISM

a E 2

+0.01

0

1 4 a

Efect of Oxygen Concentration and Carbon Monoxide on “CHC13 Metabolism

A - LIVER

-0.01 I* L

-0.02

L~.~~l....I~~~~l,.,.l

The metabolism of 14CHC13 to 14C02 and covalently bound radioactivity was reduced when the oxygen concentration was reduced from 100 to 20% in the reaction vessel (Fig. 10). A 4: 1 mixture of N2 and Oz reduced microsomal metabolism by approximately 70% for renal cortex and by 30 to 50% for liver. A 4:l mixture of CO and O2 reduced both renal cortical and hepatic microsomal metabolism to a greater extent; metabolism was reduced by approximately 90% in renal cortical and by 80% in hepatic microsomes.

350

Inhibitors of cytochrome P-450 did not affect renal cortical microsomal metabolism of 14CHC13 to 14C02 and covalently bound radioactivity in a similar manner (Fig. 11). SKF 525-A ( 1O-3 and 10e4 M) appeared to reduce the metabolism of 14CHC13 to 14C02, but did not affect covalent binding. Metympone ( 1Op4 and 10e5 M) had an opposite effect; covalent binding was decreased with no effect on 14C02 production. Piperonyl butoxide ( 1O-4 M) and a-naphthoflavone ( low4 and lop5 M) also decreased the metabolism of 14CHC13 to covalently bound radioactivity with little or no effect on 14C02 production. The same phenomenon was observed in hepatic microsomes; inhibition of 14CHC13 metabolism to 14C02 and covalently bound radioactivity was not similar (Fig. 12). Covalent binding was decreased markedly by SKF 525-A (10-j and lop4 M) and metyrapone

450

Wavdength

500

550

(nm)

+0.02 B - KIDNEY r : E 0 z 2 *

a

+0.01

0

-0.01

-0.02

Efect of Mixed Function Oxidase Inhibitors on ‘“CHC& Metabolism

400

:%L

I~~~~I~l~.l~~~,l~.,‘J 350

400

450

Wavelength

500

550

(nm)

FIG. 9. Binding spectrum of CHC13 with hepatic (A) and renal cortical (B) microsomal cytochrome P-450. Each cuvette contained 2 ml of 0.1 M sodium phosphate buffer, pH 7.0, and 2 mg microsomal protein. The baseline was recorded, and 12.5 pmol CHCl, (diluted 1:10 in ethanol) was added to the sample cuvette by a microliter syringe. An equal volume of ethanol was added to the reference cuvette. After mixing, the difference spectrum was recorded. The baseline was subtracted from the change in light absorbance caused by the addition of CHC13 to the sample cuvettes and the resultant difference spectra were plotted. Spectra were obtained at room temperature.

( 10e4 and 10e5 M); there was a tendency to decrease 14C02 but not to a statistically significant degree. Neither piperonyl butoxide ( 1Op4and 1Om5M) nor a-naphthoflavone ( 1Om4 and 10e5 M) decreased covalent binding, although there were similar decreases in 14COZ production.

520

SMITH AND HOOK

El LB

O?(100%) N2:02

(4:l)

tsi

co:o,

(4:l)

“CO>

DETECTED

(nmol I reaction

vessel )

COVALENT BINDING ( n mol I mg protein )

KIDNEY

5.0

r

2.5 r

LIVER

FIG. 10. Effect of carbon monoxide and oxygen concentration on male renal cortical and hepatic microsomal metabolism of WHCls to i4COz and covalently bound radioactivity. Reaction vessels contained 1 mg microsomal protein, 3.12 pmol ‘%HCls (specific activity 0.5 FCi/pmol, added in a volume of 2.5 ~1 of dimethylfonnamide), 0.1 pmol NADPH, 0.3 rmol NADP+, 0.3 pmol NADH, 4.5 amol glucose 6-phosphate, 1 unit glucosed-phosphate dehydrogenase, and 0.16 mmol MgCl, in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 0.75 ml. Reaction vessels were gassed for 5 min prior to adding ‘%HCls with either 100% Or, Nz:02 (4: 1 mixture), or CO:02 (4: 1 mixture). Values have been corrected for nonenzymatic metabolism of ‘%ZHCl, by incubations without a NADPH regenerating system. Values are X + SE; n = 6.

DISCUSSION A major finding of this study was that a microsomal fraction of renal cortex could metabolize CHQ and that this metabolism appeared to be mediated by cytochrome P-450. Several lines of evidence support the involvement of cytochrome P-450 in the renal metabolism of CHCls . ( 1) Significant CHCls metabolism occurred in male mouse renal cortical microsomes which contain approximately sixfold higher concentrations of cytochrome P-450 than in female mice (Smith et al., 1984). The inability to detect female renal cortical microsomal metabolism of CHCIJ was consistent with the absence of CHC13 nephrotoxicity in female mice in vivo (Smith et al., 1983) and in vitro (Smith and Hook, 1983b). (2) Renal microsomal metabolism of CHC& required the presence of oxygen and NADPH

(Figs. 7 and 10). Microsomal metabolism of CHC& was inhibited when incubations were conducted under an atmosphere of carbon monoxide (Fig. lo), which was consistent with the reduced in vitro metabolism of CHC& in male mouse renal cortical slices in the presence of carbon monoxide (Smith and Hook, 1983a,b). (3) The detection of a type I binding spectrum produced by CHC& with oxidized male renal cortical microsomes (Fig. 9) provided further evidence for the involvement of cytochrome P-450 in the renal metabolism of CHC& analogous to the role of cytochrome P-450 in hepatic metabolism of CHCl3. Differences between requirements for optimal renal and hepatic microsomal metabolism of CHC& suggested that different forms of cytochrome P-450 in mouse liver and kidney mediated CHQ metabolism. For example, NADH alone could not support me-

RENAL

14C02 (nmol Control 3.506t

CHLOROFORM

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+ l4.p

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Elhonol

r 0.516 *

100 r 80 z c s

-

m-

B 40bE 20 o-

10-3 16’ SKF

16’

10-s

10-4 10-s

16’

KP

M

525-A

KIDNEY COVALENT (nmol Control

+ “20

1.259

? 0.416

BINDING

I mg protein)

*

1 FIG. 11. Effect of cytochrome P-450 inhibitors on male renal cortical metabolism of “CHClI to 14C0, and covalently bound radioactivity. Reaction vessels contained 1 mg microsomal protein, 3.12 amol “CHC19 (specific activity 0.5 &/pmol, added in a volume of 2.5 ~1 of dimethylformamide), 0.1 amol NADPH, 0.3 pmol NADP+, 0.3 pmol NADH, 4.5 pmol glucose 6-phosphate, 1 unit glucose-6-phosphate dehydrogenase, and 0.16 mmol MgQ in 0.1 M sodium phosphate buffer, pH 7.4, in a total volume of 0.75 ml. Stock solutions of inhibitors or vehicle (Hz0 or ethanol) were added as 1% of the total reaction volume to achieve the indicated inhibitor concentrations. SKF 525-A and metyrapone were formulated in H,O, piperonyl butoxide and a-naphthoflavone were formulated in ethanol. Values have been corrected for nonenzymatic metabolism of “CHC19. Metabolism of

METABOLISM

521

tabolism of CHC& by renal cortical microsomes in contrast to the liver (Figs. 7 and 8). Furthermore, the concentration of oxygen in the incubation vessel was more critical for renal cortical than for hepatic microsomal metabolism of CHC13 (Fig. 10). These differences were emphasized further by the inhibitor studies. Dose-dependent decreases in covalent binding and 14C02 production by liver microsomes were produced by SKF 525-A and metyrapone (Fig. 12). In contrast, the effect produced by these compounds in renal microsomes showed no consistent pattern (Fig. 11) nor did the effect of piperonyl butoxide or a-naphthoflavone in either tissue. These data are consistent with hepatic but not renal metabolism of CHC13 by a phenobarbital inducible form of cytochrome P-450. The presence of 5 mM glutathione in renal cortical and hepatic microsomal reactions decreased the amount of 14CHClJ covalently bound to the trichloroacetic acid precipitable protein; there was a corresponding increase of radioactive metabolites soluble in the aqueous phase (Table 2). This increase of aqueous soluble metabolites in the presence of microsomes plus glutathione may represent the formation of a reactive intermediate, possibly phosgene which could be conjugated with glutathione to form diglutathionyl dithiocarbonate as has been described to occur in the liver (Pohl et al., 1981). Covalent binding after in vivo administration of a radiolabeled model toxicant often has been used as an indication of metabolism by and toxicity to a specific organ (Jollow et al., 1973; Ilett et al., 1973; Gillette, 1974; Boyd

“CHCl, by microsomes plus vehicle (Control + Hz0 and Control + Ethanol) was expressed as nanomoles CHC19 metabolized to 14C02 and to covalently bound radioactivity. Metabolism of “CHCls by microsomes in the presence of inhibitors was expressed as percentage of the appropriate vehicle control. Values are X If: SE; n = 5. Statistical significance was determined by calculating the 95% confidence interval for microsomal reactions containing inhibitors. *Significantly different from control + appropriate vehicle, p < 0.05.

522

SMITH AND HOOK

14C02 (nmol Contrd 4 645

120

r

100

-

a b s u %

00

-

60

-

be

40

-

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LIVER DETECTED

I reaction

vessel 1 Con,,.,,

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3.58,

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0 437

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LIVER COVALENT (nmol Conlrol

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BINDING

I mg protein) Control 1.539

+ Ethand f 0.301

120 100

FIG. 12. Effect of cytochrome P-450 inhibitors on male hepatic metabolism of “CHC13 to 14C02 and covalently bound radioactivity. Incubation conditions and data presentation are described in Fig. 1 I.

et al., 1975). In the case of CHC13, a similar degree of covalent binding to liver and kidney microsomal protein was observed after in vivo 14CHC13 administration (Ilett et al., 1973); however, previous studies could detect little or no covalent binding to renal microsomal protein compared to covalent binding to hepatic microsomal protein in vitro (Ilett et al.,

1973; Clemens et al., 1979). The data in Fig. 10 suggested that optimal metabolism of a xenobiotic by renal cortical cytochrome P-450 in vitro may require greater concentrations of oxygen than metabolism by hepatic cytochrome P-450. This observation may be similar to the situation that exists in vivo. The renal cortex receives a very high proportion of blood flow and, therefore, oxygen in relation to its mass (Maher, 1976). Furthermore, cytochrome P-450 activity in the kidney appears to be greatest in the renal cortex, particularly in the proximal tubules (Fowler et al., 1977; Zenser et al., 1978; Rush et al., 1983), the site of CHC13 toxicity. In the liver, the centrilobular region is the site of CHC& toxicity. This region of the liver contains a high proportion of a phenobarbital-inducible form of cytochrome P-450, but is believed to receive a relatively lower oxygen concentration than that delivered to the periportal region where blood first enters the liver lobules (Baron et al., 1978; James et al., 1981; Sweeney, 1981; Matsumura and Thurman, 1982). The data from these studies may suggest that specific cells within the proximal tubules can metabolize xenobiotics to a greater extent and are, therefore, more susceptible to toxicity than liver cells for certain toxicants. Traditionally, microsomal metabolism is expressed as nanomoles substrate metabolized per milligram microsomal protein. By this calculation, hepatic metabolism of 14CHC13 was approximately twice that of renal cortical “CHCls metabolism (Table 1). However, it is not surprising that more metabolism of 14CHC13was measured in hepatic microsomes since there was approximately four times more hepatic cytochrome P-450 per milligram microsomal protein than renal cytochrome P450 per milligram microsomal protein (Table 1). Thus, when metabolism was expressed as nanomoles CHC& metabolized per nanomole cytochrome P-450, there appeared to be greater renal metabolism of CHC& (Table 1). Therefore, in those kidney cells with cytochrome P-450, there may be more CHClr metabolism (and toxicity) than in liver.

RENAL

CHLOROFORM

In conclusion, these data indicate that renal cytochrome P-450 metabolizes CHC13 to a reactive intermediate.

METABOLISM

523

ILE-IT, K. F., REID, W. D., SIPES, I. G., AND KRISHNA, G. (1973). Chloroform toxicity in mice: Correlation of renal and hepatic necrosis with covalent binding of metabolites to tissue macromolecules. Exp. Mol. Pathol. 19, 2 15-229.

ACKNOWLEDGMENTS The authors thank Laura Everett, Allen Reynolds, Julie Eldredge, and Gay DeShone for excellent technical assistance and Diane Hummel and Janet Dingerdissen for preparation of the manuscript. Supported in part by USPHS Grant ES02967.

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EADIE, G. S. (1952). On the evaluation of the constants V, and KhA in enzyme reactions. Science 116, 688. ESCHENBRENNER,A. B. (1944). Induction of hepatomas in mice by repeated oral administration of chloroform, with observations on sex differences. J. Nat. Cancer Inst.

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RUSH, G. F., MAITA, K., SLEIGHT, S. D., AND HOOK, J. B. (1983). Induction of rabbit renal mixed-function oxidases by phenobarbital: Cell specific ultrastructural

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