ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 286, No. 1, April, pp. 46-56, 1991

Cytotoxicity of Alkylating Agents in Isolated Rat Kidney Proximal Tubular and Distal Tubular Cells’ Lawrence H. Lash’ and Edythe B. Woods Department

of Pharmacology,

Wayne State University,

Received September 14, 1990, and in revised form November

School of Medicine, 540 East Canfield Avenue, Detroit, Michigan

48201

14,199O

The heterogeneity of the nephron, which exhibits itself morphologically, biochemically, and functionally (1, 2),

has toxicological and pathological consequences. This is because exposure of the kidneys to specific nephrotoxicants or to conditions such as ischemia or hypoxia produces cell type-specific patterns of injury. Underlying reasons for these patterns have only been partially elucidated. In some cases, the presence of specific bioactivation enzymes or specific transport systems in one cell type and not in another can account for a chemical’s cytotoxicity. In other cases, however, the factors responsible for a given cell type’s sensitivity to a specific chemical or class of chemicals cannot be ascribed to a single difference. Rather, the biochemical and metabolic profile of the cells of interest must be examined in greater detail to discern differences that under the appropriate conditions can be responsible for marked differences in susceptibility to cellular injury. To approach this problem, we developed an in. vitro separation procedure using Percoll density-gradient centrifugation to obtain highly purified populations of cells from different regions of the rat nephron (3). With these cell populations, we can study the biochemical basis for chemical-induced injury in various regions of the nephron by assessing directly differences in effects on such properties as cellular energetics, cellular GSH status, and active transport systems. We have employed the Percoll procedure to separate isolated, renal proximal tubular (PT)3 and distal tubular (DT) cells from a mixed preparation of isolated, renal cortical cells. We have confirmed the PT cell specificity of cephaloridine-induced nephrotoxicity (3) and have characterized the susceptibility of isolated PT and DT cells to oxidative injury induced by peroxides and menadione (4). In the latter study, we found that DT cells were much more susceptible than PT cells to oxidative injury and that differences in the ability of

i This study was supported by National Institutes of Health Grant DK40725 to L.H.L. Portions of this work were presented at the 74th annual meeting of the Federation of American Societies for Experimental Biology, l-5 April, 1990, Washington DC (Woods, E. B., and Lash, L. H., 1990, FASEB J. 4, A1143). * To whom correspondence should be addressed.

3 Abbreviations used: PT, proximal tubular; DT, distal tubular; MVK, methyl vinyl ketone; AA, ally1 alcohol; NDA, N-dimethylnitrosamine; LDH, lactate dehydrogenase; BSO, buthionine sulfoximine; acivicin, L(aS,5S)-cY-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, CCCP, carbonyl cyanide m-chlorophenylhydrazone.

Patterns of chemical-induced cytotoxicity in different regions of the nephron were studied with freshly isolated proximal tubular and distal tubular cells from rat kidney. Three model alkylating agents, methyl vinyl ketone, ally1 alcohol, and N-dimethylnitrosamine, were used as test chemicals. Methyl vinyl ketone and a metabolite of ally1 alcohol, acrolein, are Michael acceptors that bind to cellular protein sulfhydryl groups and GSH. N-Dimethylnitrosamine binds to cellular protein and DNA. Lactate dehydrogenase leakage was used to assess irreversible cellular injury. Distal tubular cells were more susceptible than proximal tubular cells to injury produced by methyl vinyl ketone or ally1 alcohol while the two cell populations were equally susceptible to injury produced by Ndimethylnitrosamine. Preincubation of both proximal tubular and distal tubular cells with GSH protected them from methyl vinyl ketone- and ally1 alcohol-induced cytotoxicity but had no effect on N-dimethylnitrosamineinduced cytotoxicity. Similarly, incubation of cells with methyl vinyl ketone or ally1 alcohol, but not N-dimethylnitrosamine, altered cellular GSH status. As with GSH status, incubation of cells with methyl vinyl ketone or ally1 alcohol, but not N-dimethylnitrosamine, caused pronounced inhibitory effects on mitochondrial function, as evidenced by ATP depletion and inhibition of cellular oxygen consumption. These results demonstrate that alkylating agents are cytotoxic to both proximal tubular and distal tubular cells, and that interaction with cellular GSH is a factor determining nephron cell type specificity 0 1991 Academic Press, Inc. of injury.

46 All

Copyright 0 1991 rights of reproduction

ooo3-9861/91 $3.00 by Academic Press, Inc. in any form reserved.

NEPHROTOXICITY

OF ALKYLATING

the cells to utilize both extracellular and intracellular GSH for detoxication processes and in the ability of the cells to regenerate GSH after an oxidant challenge were contributory factors. We concluded that PT cells are better equipped than DT cells to maintain or reestablish redox homeostasis after a challenge. In the current work, we wanted to extend these findings by characterizing the susceptibility of PT and DT cells to chemicals whose cytotoxicity is associated with alkylation of cellular nucleophiles and chose three chemicals as model toxicants. Two of the chemicals, methyl vinyl ketone (MVK) and ally1 alcohol (AA), were chosen because of their reactivity with soft nucleophiles such as GSH and protein sulfhydryl groups. MVK is a Michael accentor and AA is metabolized bv renal alcohol dehvdrogknase (EC 1.1.1.1) to acrolein, which is a reactive Michael acceptor that, like MVK, has a high affinity for GSH and other sulfhydryl groups (5). Toxicity of AA and that of its metabolite acrolein have been studied previously in isolated, rat renal cortical cells (6), in a variety of in vitro liver preparations (7-15), and in uiuo (16). Toxicity of MVK has been studied in isolated, rat renal cortical cells (15, 17). The third chemical, N-dimethylnitrosamine (NDA), differs from the other two alkylating agents in that it is metabolized by cvtochrome P450 to formaldehyde and a methylating group (i.e., methyl diazonium ion) that reacts preferentially with hard nucleophiles, such as nitrogen and oxygen atoms on protein and DNA,

and

has been

studied

as a carcinogen

(18).

More-

over, little reactivity of NDA with GSH or other cellular sulfhydryl groups has been observed (19). These three chemicals have thus been chosen as probes to characterize and contrast the susceptibility of renal PT and DT cells to alkylating agents. Although the primary toxicity associated with in vivo exposure to these three chemicals is not renal, their use in this in vitro model system is relevant because of the prominence of alkylating agents as reactive metabolites associated with chemical-induced nephrotoxicity. Comparison of the effects of these three alkylating agents on cellular function and viability in PT and DT cells will provide information on the importance of alkylation as a biochemical mechanism of nephrotoxicity and on the relative importance of alkylation of hard versus soft nucleophiles in the two cell populations. MATERIALS

AND METHODS

Materials. Percoll, collagenase (type I), BSO, pyrazole, y-glutamylglutamate, MVK, and NDA were purchased from Sigma Chemical Co. (St. Louis, MO). Disulfiram and AA were purchased from Aldrich Chemical Co. (Milwaukee, WI). Acivicin [L-(aS,5S)-a-amino-3-chloro4,5-dihydro+isoxazoleacetic acid] was a gift from Dr. James Bus at the Upjohn Co. (Kalamazoo, MI). Other chemicals were of the highest purity available and were obtained from commercial sources. Isolation of rat renal PT and DT cells. Isolated renal cortical cells were prepared by collagenase perfusion (21) from male Fischer 344 rats (200 to 300 g; Charles River Laboratories, Wilmington, MA). Animals

AGENTS

47

were housed in the Wayne State University vivarium, were allowed access to laboratory chow and water ad libitum, and were kept in a room on a 12-h light-dark cycle. To obtain enriched populations of PT and DT cells, cortical cells were subjected to density-gradient centrifugation in Percoll, as previously described (3). Briefly, cortical cells (5 ml, 5 to 8 X lo6 cells/ml) were layered on 35 ml of 45% (v/v) isosmotic Percoll solution in 50-ml polycarbonate centrifuge tubes and were centrifuged at 4°C for 30 min at 20,OOOgin a Sorvall RCSB centrifuge in an SS34 rotor. The density gradient produced (1.016 to 1.120 g/ml) was continuous and concave with an inflection point at 1.057 g/ml. PT cells (1.02 to 1.05 g/ml) were identified by measurement of y-glutamyltransferase (EC 2.3.2.2) and alkaline phosphatase (EC 3.1.3.1) activities as marker enzymes and DT cells (1.08 to 1.12 g/ml) were identified by measurement of hexokinase (EC 2.7.1.1) activity as a marker enzyme (2). On the basis of these enzyme activities, the purity of the PT cell preparation was estimated to be 97% and the purity of the DT cell preparation was estimated to be 88% (3). A qualitative assessment of cell-type purity was obtained by comparing various functional responses, such as the ability of various metabolic substrates to support cellular respiration and the effects of specific inhibitors on cellular respiration (3). Before incubations, cells were diluted fivefold with Krebs-Henseleit buffer, pH 7.4, containing 25 mM Hepes, 2% (w/v) bovine serum albumin, and substrates (5 mM glucose, 5 mM glutamine), were washed to remove Percoll, and were resuspended in fresh buffer at concentrations of 1 to 4 X 10s cells/ml. Cell concentrations were determined in the presence of 0.2% (w/v) trypan blue in a hemacytometer, and cell viability was estimated by measuring the fraction of cells that excluded trypan blue or by measuring the leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) activity from the cells (21). LDH activity was measured spectrophotometrically as NADH oxidation in the presence of pyruvate; leakage was determined by comparing activity in the absence and presence of Triton X-100. The percentage of total LDH released was then determined as the ratio of LDH activity in the absence of detergent to that in the presence of detergent. Both trypan blue uptake and LDH leakage are due to damage to the plasma membrane; the data are thus taken to represent irreversible cellular injury (21). Other assessments of functional integrity were also performed, including measurement of cellular adenine nucleotide and GSH concentrations, and measurement of cellular respiration. All buffers were equilibrated with 95% 02/5% CO2 and incubations were performed at 37’C under an atmosphere of 95% 0,/5% CO2 in a Dubnoff metabolic shaking incubator (60 cycles/min). Experimental protocol. Before experiments were performed, isolated PT and DT cells were placed in 25-ml plastic Erlenmeyer flasks and were sealed with serum bottle stoppers. The atmosphere was equilibrated with 95% 02/5% COa, and the cells were preincubated for 15 min at 37’C with either Krebs-Henseleit buffer or other additions as indicated. Whenever samples were removed from the flasks, the flasks were resealed and reequilibrated with 95% 0,/5% CO*. This preincubation period served to stabilize several parameters in the cell preparations, such as adenine nucleotide and GSH concentrations, and thus provided for better cells. The 15.min preincubation time was chosen based on previous studies with freshly isolated renal cortical cells and with the purified PT and DT cell preparations (3,4,20,21). Additions to cell suspensions were made from concentrated stock solutions so that cells were minimally diluted. After the preincubation period(s), cells were incubated with either buffer (= control) or one of the three alkylating agents, each added from tenfold concentrated stock solutions in Krebs-Henseleit buffer. Concentrations of agents (0.01, 0.1, and 1 mM) and incubation times (O-2 h) were chosen for measurements of LDH release (Figs. l3) so as to produce a full range of cytotoxic responses, ranging from no significant increase in LDH release as compared with controls to nearly complete release of cellular LDH. To examine the effects of modulation of cellular GSH status on MVK-, AA-, and NDA-induced cytotoxicity (Fig. 4), cells were incubated with the three agents for 1 h before measurement of LDH release. The l-h time point was chosen because it gave significant cytotoxicity but modulation of toxicity by preincubations could still be clearly demon-

48

LASH

AND

strated. To inhibit GSH turnover, cells were preincubated for 15 min with 1 mM BSO and 0.25 mM acivicin (22, 23). Intracellular GSH concentrations were increased by preincubating cells with 5 mM GSH for an additional 30 min prior to exposure to the alkylating agents. Cells were washed and resuspended in fresh buffer before incubations were performed. There were thus four preincubation groups in these experiments: (i) buffer/buffer, (ii) buffer/GSH, (iii) (BSO + acivicin)/buffer, and (iv) (BSO + acivicin)/GSH. The preincubation with GSH increased intracellular GSH concentrations by 280% in PT cells and by 30% in DT cells (4). Regardless of the preincubation protocol used, all samples had LDH leakage values at time = 0 of 8 to 12%. Although cytotoxicity, as assessed by LDH release, was generally observed after l-h incubations, changes in concentrations of GSH or adenine nucleotides and functional effects, such as changes in rates of mitochondrial respiration, generally occurred at earlier time points if they were of primary importance in the mechanism of toxicity (3, 4, 21). For this reason, measurements of effects on cellular concentrations of GSH and GSSG (Table I) or adenine nucleotides (Tables II and III) were performed after 30-min incubations and measurements of effects on rates of cellular oxygen consumption (Fig. 5) were performed after l-h incubations. Assays. Cellular oxygen consumption was measured polarographitally with a Clark-type electrode at 37°C. The electrode was calibrated with air-saturated buffer at 37°C. Basal (i.e., no additional substrates added) and CCCP-uncoupled (equivalent to maximally stimulated rate) respiratory rates were measured. For analysis of intracellular concentrations of GSH and GSSG, cells were separated from the extracellular medium by layering 0.5 ml of cell suspension on 1 ml of 20% (v/v) Percoll in saline and centrifugation for 30 s in a microcentrifuge. Cells were then resuspended in saline and protein was removed by addition of perchloric acid. Intracellular concentrations of GSH and GSSG were measured as the S-carboxymethyl, N-dinitrophenyl and N,N-bis-dinitrophenyl derivatives, respectively, by the high-pressure liquid chromatography method of Fariss and Reed (24). Separations were achieved with a PBondapak amine lo-pm cartridge (8 mm X 10 cm) (Waters Assoc., Milford, MA) with a Waters Model 600E multisolvent delivery system using a methanol-acetate mobile phase and gradient elution. Derivatives were detected at 365 nm on a Waters Model 490 detector and were quantitated with respect to standards using a Waters Model 745 data module. Cellular adenine nucleotide concentrations were measured in neutralized perchloric acid extracts of samples by the high-pressure liquid chromatography method of Jones (25). Separation of ATP, ADP, and AMP was achieved with a PBondapak Cls lo-pm cartridge (8 mm X 10 cm) (Waters Assoc., Milford, MA) with a Waters Model 600E multisolvent delivery system. Separation conditions and sample analysis were performed as previously described (3). Data anulysis. All measurements were done on at least three separate cell preparations with paired control cells (i.e., cells incubated only with Krebs-Henseleit buffer) and are expressed as means + SE. Statistical analyses were performed with Statview SE+Graphics and SuperAnova (Abacus Concepts, Berkeley, CA) on a Macintosh SE computer. Significant differences between means for data obtained in either PT or DT cells or between means comparing data obtained in PT cells to data obtained in DT cells were first analyzed by either a one-way or a twoway analysis of variance. When significant “F-values” were obtained with the analysis of variance, the Fisher’s protected least significant difference t test was performed between relevant pairs of data to determine which means were significantly different from one another with two-tail probabilities less than 0.05 considered significant. For the analysis of data in experiments describing the time and concentration dependence of alkylating agent-induced LDH release (Figs. I-3), six types of comparisons were made. For data within a cell type, LDH release found with the following pairs was compared: (i) control cells treated with buffer at a given incubation time vs cells treated with a particular agent at a particular concentration at the same incubation time; this comparison tells which agents significantly increase LDH

WOODS release as compared with the buffer-treated controls at the corresponding incubation time; (ii) cells treated with a particular agent at a particular concentration at one incubation time vs cells treated with the same agent at the same concentration but at a different incubation time; this comparison tells the time dependence of the toxicity of a particular agent; (iii) cells treated with a particular agent at one concentration at a given incubation time vs cells treated with the same agent at a different concentration at the same incubation time; this comparison tells the concentration dependence of toxicity for a particular agent; and (iv) cells treated with a particular agent at a given concentration at a given incubation time vs cells treated with a different agent at the same concentration and incubation time; this comparison tells the relative sensitivity of a particular cell type to different agents. For data in PT and DT cells, two types of comparisons were made: (v) PT cells incubated with a particular agent at a particular concentration and incubation time vs DT cells incubated with the same agent at the same concentration and incubation time; this compares the sensitivity of the two cell types to a given agent; and (vi) PT cells incubated with a particular agent at a particular concentration and incubation time vs DT cells incubated with a different agent at the same concentration and incubation time; this compares the relative toxicity of the various agents in the two cell populations. For analysis of data in experiments examining the effect of modulation of GSH status on cytotoxicity (Fig. 4), four types of comparisons were made for data within a given cell population: (i) cells preincubated with buffer/GSH or (BSO + acivicin)/buffer or (BSO + acivicin)/GSH and incubated with a particular agent vs cells preincubated with buffer/ buffer and incubated with buffer; this comparison tells which agents within a given preincubation group are toxic; (ii) cells preincubated with buffer/buffer and incubated with either buffer or a particular agent vs cells preincubated with buffer/GSH and incubated under the same conditions; this comparison gives the effect of GSH preincubation on the cytotoxicity (i.e., LDH release) induced by a given agent; (iii) cells preincubated with buffer/buffer and incubated with either buffer or a particular agent vs cells preincubated with (BSO + acivicin)/buffer and incubated under the same conditions; this comparison gives the effect of preincubation with BSO + acivicin on the cytotoxicity of a given agent; (iv) cells preincubated with buffer/GSH and incubated with either buffer or a particular agent vs cells preincubated with (BSO + acivicin)/ GSH and incubated under the same conditions; this comparison gives the effect of BSO t acivicin on the GSH effect; and (v) cells preincubated under specific conditions and incubated with either buffer or a particular agent vs cells preincubated under the same conditions and incubated with a different agent; this compares the susceptibility of the cell population within each preincubation group to a particular agent. For data in PT and DT cells, results from the two cell populations preincubated and incubated under the same conditions were compared. For analysis of data in experiments examining the effects of the alkylating agents on cellular respiration (basal and CCCP-stimulated rates; Fig. 5), the following comparisons were made: within cell type, (i) cells incubated with a particular agent vs cells incubated with buffer, and (ii) cells incubated with a particular agent vs cells incubated with a different agent; between PT and DT cells, (iii) cells incubated with the same agent, and (iv) cells incubated with different agents. For analysis of data in experiments examining effects of the alkylating agents on cellular concentrations of GSH and GSSG (Table I) and on cellular adenine nucleotide status (ATP, ADP, AMP, ATP/ADP, and energy charge; Tables II and III), the following comparisons were made: within cell type, (i) cells incubated with a particular agent for a given incubation time vs cells incubated with the same agent for a different incubation time, (ii) cells incubated with buffer for a given incubation time vs cells incubated with a particular agent for the same incubation time, and (iii) cells incubated with a particular agent for a given incubation time vs cells incubated with a different agent for the same incubation time; between PT and DT cells, (iv) cells incubated with a particular agent for a given incubation time in both cell populations, and (v) PT cells incubated with one agent for a given incubation time

NEPHROTOXICITY

OF ALKYLATING

Tmw,hl

Timew

FIG. 1. Time and concentration dependence of methyl vinyl ketone (MVK) cytotoxicity in renal proximal tubular (PT) and distal tubular (DT) cells. Isolated renal PT (A) and DT (B) cells (2-3 X lo6 cells/ml) were incubated for the indicated times at 37°C with either Krebs-Henseleit buffer (0) or 0.01 (O), 0.1 (U), or 1 (m) mM MVK. Cytotoxicity was assessed by measurement of lactate dehydrogenase (LDH) leakage. Results are the means f SE of three cell preparations.

vs DT cells incubated time.

with a different

agent for the same incubation

RESULTS Time and concentration dependence of cytotoxicity. To obtain an initial assessment of the sensitivity of PT and DT cells to MVK, AA, and NDA, time and concentration dependences of cytotoxicity, as determined by LDH release, were measured (Figs. l-3). Although the data for each individual alkylating agent are shown separately, which allows assessment of the time and concentration dependence of the cytotoxicity of an individual agent, they were analyzed together to assess the susceptibility of each cell type to the three agents and to compare the susceptibility of the two cell types to each particular agent. A time- and concentration-dependent increase in LDH release was observed for both PT cells and DT cells incubated with either buffer or any of the three alkylating agents at the three concentrations studied. Because the control cells also exhibited higher amounts of LDH release with time, analysis of other comparisons was necessary to reveal buffer-independent effects. Comparison of LDH release data for cells incubated with any of the three agents at the three concentrations at l- and 2-h incubation times with those of buffer-treated cells incubated for 1 and 2 h demonstrated the time- and concentration-dependent cytotoxicity of these chemicals. Incubation of isolated PT cells with 1 mM MVK for 1 and 2 h, 1 mM AA for 1 and 2 h, 0.1 mM NDA for 1 h, and 1 mM NDA for 1 and 2 h produced significantly greater LDH release than cells incubated with buffer for the same times. Incubation of isolated DT cells with 0.01, 0.1, or 1 mM MVK, 0.1 or 1 mM AA, or 0.1 or 1 mM NDA for 1 and 2 h produced significantly greater LDH release than cells incubated with buffer for 1 and 2 h.

49

AGENTS

Timem,

Tim ml

FIG. 2. Time and concentration dependence of ally1 alcohol (AA) cytotoxicity in renal proximal tubular (PT) and distal tubular (DT) cells. Isolated renal PT (A) and DT (B) cells (2-3 X lo6 cells/ml) were incubated for the indicated times at 37°C with either Krebs-Henseleit buffer (0) or 0.01 (O), 0.1 (O), or 1 (m) mM AA. Cytotoxicity was assessed by measurement of lactate dehydrogenase (LDH) leakage. Results are the means f SE of three cell preparations.

Concentration-dependent differences in amounts of LDH release were also observed. In isolated PT cells, concentration-dependent increases in LDH release occurred in cells incubated with MVK (0.01 mM vs 1 mM at 1 and 2 h; 0.1 mM vs 1 mM at 1 and 2 h), AA (0.01 mM vs 1 mM at 1 and 2 h; 0.01 mM vs 0.1 mM at 2 h), and NDA (0.01 mM vs 1 mM at 1 and 2 h; 0.1 mM vs 1 mM at 2 h). In isolated DT cells, concentration-dependent increases in LDH release occurred in cells incubated with MVK (0.01 mM vs 0.1 mM and 1 mM at 1 and 2 h; 0.1 mM vs 1 mM at 1 and 2 h) and AA (1 mM vs 0.1 InM and 0.01 mM at 2 h); the cytotoxicity of NDA did not exhibit any concentration dependence in DT cells.

Time ,h,

Tme ,h)

FIG. 3. Time and concentration dependence of N-dimethylnitrosamine (NDA) cytotoxicity in renal proximal tubular (PT) and distal tubular (DT) cells. Isolated renal PT (A) and DT (B) cells (2-3 X lo6 cells/ml) were incubated for the indicated times at 37°C with either Krebs-Henseleit buffer (0) or 0.01 (O), 0.1 (O), or 1 (m) mM NDA. Cytotoxicity was assessed by measurement of lactate dehydrogenase (LDH) leakage. Results are the means f SE of three cell preparations.

50 A

80

LASH

AND

II

Buffer

WOODS

GSH

1 6C

I-

-Buffer

BSO + Acivicin

Buffer

BSO + Acivicin

FIG. 4. Effects of modulation of GSH status on cytotoxicity of alkylating agents. Isolated renal proximal tubular (PT) (A) and distal tubular (DT) (B) cells (2-3 X lo6 cells/ml) were preincubated for 15 min with either Krebs-Henseleit buffer or 1 mM buthionine sulfoximine (BSO) + 0.25 mM acivicin (to inhibit GSH synthesis and degradation, respectively). These two groups (indicated in brackets) were subdivided further and were preincubated for an additional 30 min with either buffer (open bars) or with 5 mM GSH (filled bars). At time = 0 min, each preincubation group was incubated for 1 h with either buffer, 1 mM methyl vinyl ketone (MVK), 1 mM ally1 alcohol (AA), or 1 mM N-dimethylnitrosamine (NDA). Cytotoxicity was assessed by measurement of lactate dehydrogenase (LDH) leakage. Results are the means f SE of three to nine cell preparations. Comparison of the amount of LDH released by different agents at the same concentrations at the same incubation times, in order to assess relative sensitivity of the cell to the various agents, showed no differences in PT cells but showed marked differences in DT cells. MVK produced greater LDH release than AA and NDA at all three concentrations and in most cases, at both l- and 2-h incubation times. Although the LDH release following incubation with AA and NDA were identical for most points, 1 mM AA produced greater LDH release than NDA after 2 h incubation. Therefore, MVK was markedly more cytotoxic to DT cells than either AA or NDA and AA was significantly, but only slightly, more cytotoxic than NDA to DT cells. To assess the relative sensitivities of PT and DT cells more directly, comparisons of the amounts of LDH released from the two cell populations by the same maneuvers were made. For this approach to be valid, LDH release from PT cells and DT cells incubated with buffer for the same amounts of time must not differ significantly. This was indeed the case at all incubation times examined, thus permitting direct comparisons between responses of PT and DT cells. MVK produced greater LDH release

from DT cells than from PT cells at all three concentrations and at both time points. AA produced greater LDH release from DT cells than from PT cells only at the highest concentration (1 InM) and the longest incubation time (2 h) tested. In contrast, no significant differences were observed in the ability of NDA to cause LDH release in the two cell populations. GSH homeostasis. To characterize the role of differences in cellular redox status in susceptibility to alkylating agents, we subjected the isolated PT and DT cells to manipulations that altered cellular concentrations of GSH and examined effects on MVK-, AA-, and NDA-induced LDH leakage after l-h incubations (Fig. 4). Incubation of isolated PT cells with either 1 mM MVK, AA, or NDA produced significant increases in LDH release in those cells that were preincubated with either buffer/buffer or (BSO + acivicin)/buffer as compared with controls that were exposed to the same preincubation conditions. Preincubation with BSO + acivicin alone [buffer/buffer vs (BSO + acivicin)/buffer] had little effect on PT cells, although LDH release induced by incubation with NDA was significantly lower in the (BSO + acivicin)-pretreated cells. For the PT cells that were preincubated with buffer/

NEPHROTOXICITY

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GSH, only incubation with NDA produced a significant increase in LDH release. For the PT cells that were preincubated with (BSO + acivicin)/GSH, incubation with MVK or AA, but not NDA, increased LDH release as compared with cells incubated with buffer. Direct comparison of the buffer/buffer preincubation group with the buffer/GSH preincubation group in PT cells showed that GSH protected completely against the cytotoxicity caused by MVK and AA and partially against the cytotoxicity caused by NDA. Direct comparison of the (BSO + acivicin)/buffer and (BSO + acivicin)/GSH groups, however, showed no significant differences in LDH release. Direct comparison of the buffer/GSH preincubation group with the (BSO + acivicin)/GSH preincubation group showed that inhibition of GSH metabolism eliminated protection by exogenously added GSH with all three alkylating agents. Data for each agent within the four preincubation groups were compared to assess the relative susceptibility of the PT cells to the three alkylating agents. In the buffer/buffer preincubation group, LDH release produced by MVK and NDA was greater than that produced by AA. In the buffer/GSH preincubation group, LDH release produced by NDA was greater than that produced by either MVK or AA. In the (BSO + acivicin)/buffer preincubation group, LDH release produced by either MVK, AA, or NDA did not significantly differ from one another. In the (BSO + acivicin)/GSH preincubation group, LDH release produced by MVK was greater than that produced by AA or NDA. Modulation of GSH status in isolated DT cells had results generally similar to those described above for PT cells. All three agents produced significant increases in LDH release compared to buffer-incubated DT cells for the buffer/buffer, (BSO + acivicin)/buffer, and (BSO + acivicin)/GSH preincubation groups. In the buffer/ GSH preincubation group, only MVK and NDA produced significant LDH release compared to cells incubated with buffer. Comparison of LDH release data in the buffer/ buffer preincubation group with those in the buffer/GSH preincubation group showed that GSH completely protected DT cells from AA, as was found in PT cells, but only partially protected DT cells from MVK and had no significant effect on cytotoxicity produced by NDA. As was found in PT cells, preincubation with (BSO + acivicin) completely eliminated the protection by GSH against MVK- and AA-induced cytotoxicity. Data for each agent within the four preincubation groups were compared to assess the relative susceptibility of the DT cells to the three alkylating agents. In the buffer/buffer preincubation group, LDH release produced by MVK was greater than that produced by either AA or NDA. In the buffer/GSH preincubation group, LDH release produced by NDA was greater than that produced by either AA or MVK and LDH release produced by MVK was greater than that produced by AA; this is consistent with the partial protection by GSH against MVK- and AA-induced cytotox-

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51

icity. In both the (BSO + acivicin)/buffer and (BSO + acivicin)/GSH preincubation groups, LDH release produced by MVK was greater than that produced by either AA or NDA. Comparisons of incubations under corresponding conditions in PT cells and DT cells only showed a significant difference with MVK. In both the buffer/GSH and the (BSO + acivicin)/buffer preincubation groups, LDH release produced by incubation with MVK was greater in DT cells than in PT cells. To study further the role of cellular GSH status in the cytotoxicity of these alkylating agents, isolated PT and DT cells were incubated for 30 min with the three agents and cellular concentrations of GSH and GSSG were measured (Table I). In PT cells, only incubation with MVK significantly decreased cellular GSH concentration. In DT cells, incubation with 1 mM MVK decreased cellular GSH concentration by 40%, but this change was not significant (P = 0.08). Examination of cellular GSSG concentrations gave more prominent effects. In PT cells, GSSG concentrations were significantly higher at 30 min as compared with the 0-min results under all incubation conditions including controls. Thus, none of the agent-induced effects were independent of the buffer effect. In DT cells, however, incubation with AA significantly increased GSSG concentrations at 30 min relative to both buffer and NDA. Both AA and MVK produced significantly higher GSSG concentrations in DT cells than in PT cells after 30-min incubations. Cellular energetics. Effects of MVK, AA, and NDA on mitochondrial function were assessed first by measuring cellular concentrations of adenine nucleotides after 3Omin incubations (Tables II and III). On the basis of adenine nucleotide status, some deterioration of cellular function over the initial 30-min incubation period occurred in buffer-treated controls from both cell populations. In PT cells, no significant changes occurred in cellular concentrations of ATP, ADP, or AMP, or in the ATP/ADP ratio over the 30-min control incubation. However, a small, but significant decrease in cellular energy charge occurred in the PT control group. In DT cells, a significant decrease of nearly 50% in the cellular ATP concentration occurred in the buffer-treated controls. Although no significant changes occurred in the cellular concentrations of ADP and AMP, the cellular ATP/ADP ratio and energy charge were significantly lower after the 30-min control incubation. With the appropriate statistical analyses (see Materials and Methods section), however, effects due specifically to the alkylating agents were identified. Incubation of isolated PT cells with MVK, at both 0.1 and 1 mM concentrations, had significant effects on adenine nucleotide status, reducing cellular ATP concentrations by 35 and 50%, respectively, as compared with buffer-incubated control cells. MVK, at 1 mM, signifi-

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TABLE Effects

of Alkylating

Agents

Proximal

I

of Cellular

tubular

Distal tubular cells

GSSG (nmol/lOs

Buffer 0 min 30 min 1 mM ally1 alcohol 0 min 30 min 1 mM methyl vinyl ketone 0 min 30 min 1 mM N-dimethylnitrosamine 0 min 30 min

Homeostasis

cells

GSH Addition

GSH

GSH

cells)

GSSG (nmol/lOs

cells)

9.88 + 1.38 8.90 + 1.17

0.80 + 0.12 2.17 + 0.29

11.8 k 1.5 11.2 k 0.8

1.24 f 0.21 2.29 f 0.32

8.62 f 1.11 9.21 + 1.42

0.72 + 0.07 2.29 * 0.41

13.6 f 2.4 8.84 f 2.63

1.19 f 0.20 3.30 -+ 0.68

8.51 + 2.85 4.99 f 0.90

0.64 2 0.09 1.54 + 0.22

10.5 * 1.5 6.75 f 1.78

1.15 f 0.27 2.66 5~0.59

10.4 f 3.4 9.25 f 1.09

0.81 +- 0.15 2.13 + 0.17

12.4 + 2.9 11.4 + 2.4

1.72 + 0.28 2.30 + 0.24

Note. Isolated proximal tubular (PT) and distal tubular (DT) cells from rat kidney cortex were incubated at 37°C under an atmosphere of 95% Oz, 5% COB at concentrations of 2 to 3 X 10s cells/ml with the indicated additions. After 0- and 30-min incubations, aliquots were withdrawn for analysis of GSH and GSSG. Samples were derivatized with iodoacetic acid and l-fluoro-2,4-dinitrobenzene and were analyzed by HPLC. Results are the means + SE of samples from five cell preparations.

cantly increased cellular ADP and AMP concentrations. Of particular note was the nearly twofold increase in cellular AMP concentrations with 1 mM MVK. Cellular ATP/ADP ratios were significantly lower in PT cells incubated for 30 min with either 0.1 mM or 1 mM MVK as compared with controls and the cellular energy charge was decreased after incubation with 1 mM MVK. Incubation of isolated DT cells with 1 mM MVK for 30 min

TABLE Effects

of Alkylating

Addition Buffer 0 min 30 min 0.1 mM methyl vinyl ketone 0 min 30 min 1 mM methyl vinyl ketone 0 min 30 min 1 mM ally1 alcohol 0 min 30 min 1 mM N-dimethylnitrosamine 0 min 30 min

produced significant decreases in cellular ATP concentrations and marked changes in cellular concentrations of ADP and AMP. These effects were evident in the significant decreases in cellular ATP/ADP ratios and energy charge with both low and high MVK concentrations. In spite of the cytotoxicity of AA in both PT and DT cells (Fig. 2), no significant effects on adenine nucleotide status were observed in either cell population. Similarly,

II

Agents of Cellular Adenine Nucleotides

in Isolated Proximal

Tubular

Cells Energy charge

ATP

ADP (nmol/lOe cells)

AMP

5.19 + 0.77 4.03 + 0.69

1.42 f 0.48 1.52 f 0.44

1.05 f 0.93 1.44 f 0.32

7.38 f 1.94 3.48 + 0.51

0.80 + 0.03 0.70 f 0.02

5.35 ZIZ1.81 2.62 2 0.88

0.92 + 0.02 1.39 f 0.51

0.95 f 0.02 1.18 f 0.15

5.82 + 2.02 1.55 f 0.51

0.77 f 0.06 0.61 f 0.10

5.52 f 1.56 2.02 f 0.71

2.10 f 0.82 4.29 f 2.40

1.18 f 0.32 2.59 f 0.98

4.63 + 1.82 1.05 f 0.60

0.78 IL 0.02 0.52 f 0.01

5.63 f 1.79 3.14 f 1.32

1.08 2 0.59 1.12 f 0.57

0.77 + 0.19 1.45 * 0.77

7.38 + 4.26 3.02 f 0.82

0.82 + 0.04 0.61 f 0.10

ATP/ADP

5.33 f 1.00 3.17 f 1.15

Note. Isolated proximal tubular (PT) cells from rat kidney cortex were incubated at 37’C under an atmosphere of 95% O,, 5% COr at concentrations of 2 to 3 X 10s cells/ml with the indicated additions. After 0- and 30-min incubations, aliquots were withdrawn for analysis of ATP, ADP, and AMP by HPLC. Results are the means f SE of samples from three to six cell preparations.

NEPHROTOXICITY

OF ALKYLATING TABLE

Effects

of Alkylating

Agents

Adenine

III Nucleotides

in Isolated

Distal

Tubular

Cells Energy charge

ATP

ADP (nmol/lOs cells)

AMP

10.6 f 2.2 5.49 f 1.03

2.09 If: 0.85 2.55 f 0.73

2.75 f 0.68 2.08 zk 0.48

8.56 + 1.59 3.07 f 0.84

0.75 f 0.02 0.65 +- 0.04

8.87 f 3.76 3.32 f 0.82

0.97 + 0.08 5.68 + 2.52

2.48 + 0.73 4.47 f 2.77

9.19 f 3.65 0.68 f 0.12

0.74 + 0.03 0.48 f 0.11

11.3 f 3.2 1.82 f 0.48

2.76 f 0.80 3.58 f 0.74

2.10 + 0.61 6.05 2 1.79

4.59 IL 0.60 0.60 + 0.19

0.79 f 0.02 0.32 iz 0.03

9.80 f 3.31 5.52 + 1.41

0.88 f 0.38 2.93 f 1.47

1.57 f 0.56

Addition Buffer 0 min 30 min 0.1 mM methyl vinyl ketone 0 min 30 min 1 mM methyl vinyl ketone 0 min 30 min 1 mM ally1 alcohol 0 min 30 min 1 mM N-dimethylnitrosamine 0 min 30 min

of Cellular

53

AGENTS

ATP/ADP

1.15 f 0.58

11.3 + 1.5 4.83 k 3.62

0.80 + 0.14 0.71 f 0.07

9.77 k 1.84 4.79 + 1.50

Note. Isolated distal tubular (DT) cells from rat kidney cortex were incubated at 37°C under an atmosphere of 95% 02, 5% CO, at concentrations of 2 to 3 X 10s cells/ml with the indicated additions. After 0- and 30-min incubations, aliquots were withdrawn for analysis of ATP, ADP, and AMP by HPLC. Results are the means f SE of samples from three to six cell preparations.

NDA, although producing appreciable cytotoxicity in both cell populations (Fig. 3), did not significantly lower cellular ATP concentrations. With NDA as the test compound, only ATP could be measured because of interference with detection on the chromatogram.

Both basal and CCCP-uncoupled respiratory rates were measured after l-h incubations to assess further effects on mitochondrial function (Fig. 5). No consistent effects on basal respiratorv rates were observed. Addition of 25 PM CCCP stimulated respiration by nearly 300% in PT

0 BZQI

A

CCCF-stimulated

BUfiCX

1 mM MVK

mM AA

Treatment

1 mM NDA

40

Buffer

1mMMVK

1mMAA

1mMNDA

Treatment

FIG. 5. Effects of alkylating agents on cellular respiratory function. Isolated renal proximal tubular (PT) (A) and distal tubular (DT) (B) cells (2-3 X 10s cells/ml) were incubated for 1 h at 37°C with either Krebs-Henseleit buffer, 1 mM methyl vinyl ketone (MVK), 1 mM ally1 alcohol (AA), or 1 mM N-dimethylnitrosamine (NDA). Respiratory rate was measured polarographically as oxygen consumption as described under Materials and Methods. Both basal (i.e., no further addition; open bars) and uncoupler-stimulated [i.e., after addition of 25 pM carbonyl cyanide m-chlorophenylhydrazone (CCCP); filled bars] rates were measured. Results are the means f SE of five to nine cell preparations.

54

LASH

AND

cells and by approximately 50% in DT cells, as shown previously (3). In contrast to the minimal effects of the alkylating agents on basal respiration, incubation of either PT cells or DT cells with these chemicals had significant effects on respiratory function. In both cell populations, incubation with any of the three alkylating agents for 1 h produced nearly complete elimination of the CCCPdependent increase in the rate of oxygen consumption as respiratory rates in the presence of CCCP in each case were no longer significantly higher than basal rates. Direct comparison of respiratory rates in the presence of CCCP in PT cells incubated with one of the alkylating agents with those in cells incubated with buffer indicated that only AA produced a significant decrease. In DT cells, only MVK produced a significant decrease in the rate of CCCPstimulated respiration as compared with cells incubated with buffer. DISCUSSION

The in vitro procedure employed in this study enables us to characterize the biochemistry and function of the nephron in a well-defined, viable system. Using Percoll density-gradient centrifugation, we can prepare isolated cells from individual regions of the nephron and thereby study how each region responds to a chemical or functional challenge and assess the role of various biochemical and physiological factors in the cytotoxicity of specific chemicals or classes of chemicals (3). In a previous study (4), we investigated the cytotoxicity of chemicals that act by generating an oxidative stress. We assessed directly the function of the GSH redox system in isolated PT and DT cells in the detoxication of reactive prooxidant chemicals and demonstrated that the capacity of the two cell populations to respond to an oxidant challenge is markedly different, with the DT cells being much more susceptible to this type of injury. In the current work, we chose alkylating agents as model cytotoxic chemicals to investigate if chemicals that act primarily by covalent binding produce toxicity in isolated kidney cells, and we compared the susceptibility of PT and DT cells to this form of injury. Additionally, two different types of alkylating agents were chosen, one type, illustrated by NDA, that binds selectively to hard nucleophilic groups on protein and nucleic acid, and one type, illustrated by Michael acceptors (MVK and AA), that binds to soft nucleophilic groups such as sulfhydryl groups. This approach allowed us to extend our previous studies on susceptibility to oxidative stress to gain a more complete understanding of the various factors that determine susceptibility to chemical-induced injury in different regions of the nephron. Previous studies on NDA have generally focussed on the chemical mechanism of its binding to nucleic acids and on its ability to produce tumors, which are observed most commonly in liver and kidney, but also in other

WOODS

tissues (18). Nitrosamines such as NDA occur in the environment and can be formed in the body from ingested nitrites and nitrates. NDA can undergo cytochrome P450dependent dealkylation to generate formaldehyde and a potent methylating species or it may undergo denitrosation to form formaldehyde and monomethylamine, which is metabolically inert (26). The dealkylation reaction therefore represents a bioactivation pathway while the denitrosation reaction represents a detoxication pathway for NDA. The similar susceptibility of PT and DT cells to NDA-induced cytotoxicity (Fig. 3) indicates that the bioactivation pathway is present in both cell populations. What is not clear from these results is the degree to which differences in effects of covalent binding or in activities of the denitrosation pathway in the two cell populations account for the toxicity that was observed. The possible role of formaldehyde formation in the cytotoxicity of NDA is also unclear. To address this question, we incubated isolated PT and DT cells for up to 2 h with 0.01, 0.1, and 1 mM formaldehyde and examined effects on LDH release (data not shown). No effects were observed after either 1 or 2 h incubation in PT cells at any of the three concentrations. In DT cells, however, 0.1 and 1 mM formaldehyde increased LDH release as compared with cells incubated with buffer at 1 h (46.6 and 51.2%, respectively, vs 37.8%) and 2 h (68.8 and 7&O%, respectively, vs 53.3%), indicating that formation of formaldehyde may contribute to NDA-induced cytotoxicity in DT cells. Consideration of the amount of LDH release produced and the concentration of formaldehyde required to produce it as compared with these effects as seen with NDA, indicates that formaldehyde probably does not play a major role in the cytotoxicity observed with NDA. Therefore, although covalent binding of NDA metabolites was not measured in this study, these data and previous knowledge of NDA metabolism and NDAinduced biochemical effects (18, 26) indicate that the probable cytotoxic metabolite of NDA is the methylating species and the probable mechanism of cytotoxicity involves covalent binding to cellular nucleophiles. Acrolein is a highly reactive a&unsaturated aldehyde (Michael acceptor) that produces both hepatotoxicity (716) and nephrotoxicity in isolated, rat renal cortical cells (6). Acrolein is an environmental pollutant, is a metabolite of the chemotherapeutic agent cyclophosphamide, and is the oxidation product of AA (27). AA is metabolized by renal cytosolic alcohol dehydrogenase to acrolein. Acrolein is then metabolized by aldehyde dehydrogenase, which is found in renal cytosol and mitochondria, to the nontoxic acrylic acid (6). We confirmed in the present study that this pathway for AA metabolism is active in both PT and DT cells by examining the effects of pyrazole and disulfiram on AA-induced cytotoxicity (data not shown). Preincubation of cells with 2 mM pyrazole, which inhibits alcohol dehydrogenase, significantly diminished AA-induced LDH release, indicating that the product of this

reaction, acrolein, or a subsequent metabolite, is the toxic species. Furthermore, preincubation of cells with 0.2 mM disulfiram, which inhibits aldehyde dehydrogenase (EC 1.2.1.3), significantly increased AA-induced LDH release, confirming that acrolein is the toxic metabolite of AA and that metabolism of acrolein by aldehyde dehydrogenase to acrylic acid is a detoxication pathway. Hjelle et al. (28) found that aldehyde dehydrogenase is present in very high amounts in the pars recta of the rabbit proximal tubule and suggested that this enzyme may play an important role in detoxication of potentially cytotoxic aldehydes. A similar detoxication role for aldehyde dehydrogenase has been proposed in isolated liver cells (14, 16). Although GSH conjugation has also been considered a detoxication pathway for acrolein (6, 7), Mitchell and Petersen (29) proposed recently that the GSH-acrolein adduct can undergo reductive and oxidative metabolism in rat liver and therefore may play a role in cytotoxicity due to acrolein, and presumably due to AA as well. Data presented in this study are consistent with GSH conjugation having some role in AA and acrolein metabolism. While significant GSH depletion due to AA occurred in the DT cells, no GSH depletion due to AA was observed in the PT cells (Table I), indicating that the mechanism of cytotoxicity involved other factors in addition to GSH status. AA may not deplete PT cell GSH because its reactive metabolite acrolein may interact preferentially with other nucleophilic targets or the pathways of AA and acrolein metabolism in PT and DT cells may differ. AA-induced cytotoxicity was significantly greater in DT cells than in PT cells, although this difference only became apparent at the highest concentration and longest incubation time tested (1 mM at 2 h). Both alcohol and aldehyde dehydrogenase activities are higher in PT cells than in DT cells (28). Because the first enzyme plays a bioactivation role and the second plays a detoxication role, the balance between the two activities will be important in determining the amount of cytotoxic metabolite formed. Two additional factors in determining the susceptibility of a given cell population to AA are the availability of target sites and the activities of general detoxication pathways. Whereas the first factor, availability of target sites, is difficult to quantitate, the second factor is not. In a previous study characterizing the sensitivity of isolated PT and DT cells to oxidative stress induced by peroxides and quinones (4), we showed that DT cells were significantly more susceptible than PT cells to this form of injury, and furthermore, that a major contributing factor to this difference was cellular GSH status. Because of the interaction of acrolein with soft nucleophiles such as GSH and protein sulfhydryl groups (6-16), differences in cellular GSH status may contribute to the observed pattern of toxicity of AA in this study. The results indicate, however, that cellular GSH status may only play a significant role in the DT cells.

MVK is similarly a reactive @-unsaturated carbonyl compound (Michael acceptor) and shows a preference for reacting with GSH and other soft nucleophiles (5). MVKinduced cytotoxicity was also greater in DT cells than in PT cells. In this case, the difference between the two cell populations was more pronounced than for AA in that it was statistically significant at lower concentrations and at earlier incubation times (Figs. 1 and 2). The more rapid onset of toxicity of MVK as compared with AA is consistent with the requirement for metabolism for AA and the direct reactivity of MVK. The difference between MVK and AA in the pattern of GSH depletion may therefore be explained by either the chemistry of the reactive species involved or by the fact that MVK does not require bioactivation whereas AA must first be metabolized to acrolein. The site within the cell where AA metabolism occurs may play an important role in determining the pattern and extent of AA-induced cytotoxicity. Because the chemical reactivities of MVK and acrolein are similar (5), the site of metabolism and exposure may be the more important factor. A similar conclusion about the role of chemical reactivity as compared with the site of bioactivation may be made concerning NDA-induced cytotoxicity. In this case, both the chemistry of and the subcellular site of formation of the reactive species may be important in the mechanism of action. NDA metabolism occurs on the endoplasmic reticulum, the site of cytochrome P450 (18). The chemical reactivity of the methylating agent may preclude any direct effects on enzymes or processes at other sites within the cell. Functional changes in the cell that occur in other subcellular regions, such as the mitochondria, may therefore be secondary effects that are consequences of the primary lesion in the endoplasmic reticulum. The influence of reactive species chemistry and bioactivation site in the mechanism of cytotoxicity is illustrated by the observed changes in mitochondrial function. Using adenine nucleotide status to assess mitochondrial function (Tables II and III), we found that MVK was more toxic than either AA or NDA in the two cell populations. Using CCCP-stimulated respiration to assess mitochondrial function, however, we found somewhat different results. In PT cells, AA produced significantly more inhibition than either MVK or NDA; in DT cells, MVK produced the most inhibition of the three agents. A possible explanation for this difference in behavior is that in PT cells, more acrolein may be generated from AA within the mitochondria than is formed in DT cells and PT cells may contain better defenses against MVK-induced cytotoxicity than DT cells (4). Furthermore, in DT cells, less AA may be metabolized to its reactive metabolite and fewer defenses may be available to counteract the cytotoxicity produced by MVK. The lack of a close correlation between respiratory rate and adenine nucleotide status may indicate that one effect of these alkylating agents is to alter the regulation of cel-

56

LASH

AND

lular energetics and thus the integration of respiration and energy-dependent cellular functions. As stated above, the lack of correlation between mitochondrial effects and cytotoxicity (i.e., LDH release) indicates that for these three alkylating agents, unlike previous results with chemicals that directly produce oxidative stress (4), inhibition of mitochondrial function is not a primary mechanism of cytotoxicity but, rather, is a consequence of the cytotoxicity. For MVK and AA, but not for NDA, alterations in cellular GSH status play a role in determination of cytotoxicity. This was illustrated by the greater sensitivity of DT cells to these chemicals and by the ability of increased intracellular GSH to protect cells from the cytotoxicity. The ability of BSO and acivicin to eliminate this protection indicates that GSH must be degraded and presumably resynthesized to exert its effect. Alterations in cellular GSH status are more important for MVK-induced cytotoxicity than for AA-induced cytotoxicity. In conclusion, this study has provided information on how isolated PT and DT cells differ in their response to alkylating agents. The cytotoxicity of these alkylating agents was demonstrated and information on some of the key factors that determine their nephrotoxicity was presented. Consistent with previous results on susceptibility to oxidative stress (4), we showed that alkylating agents that can interact directly with the GSH redox system were more toxic to DT cells than to PT cells. Other modifying factors are whether or not metabolism is required for toxicity and the subcellular localization of bioactivation and detoxication steps. Alkylation of critical nucleophilic groups in isolated kidney cells is thus associated with potent cytotoxicity. The chemical nature of these groups determines the susceptibility to injury. Additional work is required to delineate more completely the mechanism of cytotoxicity due to alkylation. Mitochondrial dysfunction, although important in the development of the cytotoxic response, is a secondary effect of a primary lesion produced by these alkylating agents. REFERENCES 1. Walker, 219.

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2. Guder, W. G., and Ross, B. D. (1984) Kidney Znt. 26, 101-111. 3. Lash, L. H., and Tokarz, J. J. (1989) Anal. Biochem. 182,271-279.

WOODS 4. Lash, L. H., and Tokarz, J. J. (1990) Am. J. Physiol. 259, F338F347. 5. Estabauer, H., Zollner, H., and Scholz, N. (1975) 2. Naturforsch. 30,466-473. 6. Ohno, Y., Jones, T. W., and Ormstad, K. (1985) C&m.-Bill. Interact. 52, 289-299. 7. Ohno, Y., Ormstad, K., Ross, D., and Orrenius, S. (1985) Z’oxicol. Appl. Pharmacol. 78, 169-179. 8. Belinsky, S. A., Badr, M. Z., Kauffman, F. C., and Thurman, R. G. (1986) J. Pharmacol. Exp. 2%~. 238, 1132-1137. 9. Badr, M. Z., Belinsky, S. A., Kauffman, F. C., and Thurman, R. G. (1986) J. Pharmacol. Exp. Ther. 238,1138-1142. 10. Ku, R. H., and Billings, R. E. (1986) Arch. Biochem. Biophys. 247, 183-189. 11. Dogterom, P., Nagelkerke, J. F., van Steveninck, J., and Mulder, G. J. (1988) Chem.-Biol. Interact. 66, 251-265. 12. Penttila, K. E. (1988) Chem.-Biol. Interact. 66, 107-121. 13. Dogterom, P., Kroese, E. D., Mulder, G. J., and Nagelkerke, J. F. (1989) Biochem. Pharmacol. 38,4225-4230. 14. Silva, J. M., and O’Brien, P. J. (1989) Arch. Biachem. Biophys. 275, 551-558. 15. Zollner, H. (1973) Biochem. Pharmacol. 22,1171-1178. C., and Wendel, A. (1987) Biochem. 16. Jaeschke, H., Kleinwaechter, Pharmacol. 36, 51-57. D., and Anders, 17. Lash, L. H., Elfarra, A. A., Rakiewicz-Nemeth, M. W. (1990) Arch. Biochem. Biophys. 276, 322-330. of Foreign 18. Archer, M. C., and Labuc, G. E. (1985) in Bioactivation Compounds (Anders, M. W., Ed.), pp. 403-431, Academic Press, Orlando, FL. 19. Craddock, V. M. (1965) Biochem. J. 94,323-330. 20. Jones, D. P., Sundby, G.-B., Ormstad, K., and Orrenius, S. (1979) Biochem. Pharmacol. 28, 929-935. 21. Lash, L. H. (1989) in In Vitro Toxicology: Model Systems and Methods (McQueen, C. A., Ed.), pp. 231-262, The Telford Press, Caldwell, NJ. 22. Griffith, 0. W., and Meister, A. (1979) J. Biol. Chem. 264, 75587560. 23. Reed, D. J., Ellis, W. W., and Meek, R. A. (1980) Biochem. Biophys. Res. Commun. 94, 1273-1277. 24. Fariss, M. W., and Reed, D. J. (1987) in Methods in Enzymology (Jakoby, W. B., and Griffith, 0. W., Eds.), Vol. 143, pp. 101-109, Academic Press, Orlando. 25. Jones, D. P. (1981) J. Chromatogr. 225, 446-449. 26. Streeter, A. J., Nims, R. W., Sheffels, P. R., Heur, Y.-H., Yang, C. S., Mica, B. A., Gombar, C. T., and Keefer, L. K. (1990) Cancer Res. 50, 1144-1150. 27. Ohno, Y., and Ormstad, K. (1985) Arch. Toxicol. 67,99-103. 28. Hjelle, J. T., Petersen, D. R., and Hjelle, J. J. (1983) J. Phurmacol. Exp. Z’her. 224,699-706. 29. Mitchell, D. Y., and Petersen, D. R. (1989) J. Pharmacol. Exp. Thu.

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