Cytotoxicity of trichloroethylene andS-(1,2-dichlorovinyl)-L-cysteine in primary cultures of rat
renal proximal tubular and distal tubular cells
Brian S. Cummings a,1, Richard C. Zangar b,2, Raymond F. Novak b,Lawrence H. Lash a,*
a Department of Pharmacology, Wayne State Uni6ersity School of Medicine, 540 East Canfield A6enue, Detroit, MI 48201, USAb Institute of Chemical Toxicology, Wayne State Uni6ersity, Detroit, MI 48201, USA
* Corresponding author. Tel.: +1-313-5770475; fax: +1-313-5776739.E-mail address: [email protected] (L.H. Lash).1 Present address: University of Arkansas Medical Sciences, Division of Pharmacology & Toxicology, Slot 638, 4301 Markham
Street, Little Rock, AR 72205, USA.2 Present address: Department of Molecular Biosciences, Pacific Northwest National Laboratory, P.O. Box 999, P7-56, Richland,
B.S. Cummings et al. / Toxicology 150 (2000) 83–9884
1. Introduction
Trichloroethylene (TRI) is a colorless, volatileliquid and alkenyl halide (Manahan, 1992). Thelipophilic character, non-flammability, and highboiling point of TRI make it useful in a variety ofindustrial processes, including metal degreasingand dry cleaning, and TRI was also once used asan anesthetic (Davidson and Beliles, 1991). Be-cause of these uses, high amounts of TRI haveevaporated into the atmosphere and contaminateground and surface water and food, raising thepossibility of both occupational and general expo-sure of humans to TRI. The symptoms of toxicTRI exposure include central nervous system de-pression, abnormal liver function and irritatingeffects to the skin and mucous membranes of therespiratory tract (Reichert, 1983). TRI is also aputative human carcinogen and causes kidneycancer in male rats and possibly in humans(Davidson and Beliles, 1991; Henschler et al.,1995).
TRI is metabolized by two separate pathways.Oxidative metabolism of TRI by cytochromeP450 (P450) results in the formation of chloralhydrate, which can be further metabolized totrichloroacetic acid, dichloroacetic acid,trichloroethanol, monochloroacetic acid, and ox-alic acid (Miller and Guengerich, 1983). TRI canalso be metabolized by conjugation with glu-tathione (GSH) to form S-(1,2-dichlorovinyl)glutathione (DCVG). DCVG can befurther metabolized by g-glutamyltransferase(GGT; EC 2.3.2.1) and cysteinylglycine dipepti-dase (EC 3.4.13.12) to form S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC can be eitherN-acetylated to form N-acetyl-DCVC, which canbe deacetylated back to DCVC, or metabolized bycysteine-conjugate b-lyase (b-lyase; EC 4.4.1.13)to form a reactive thiol. The renal effects of TRIhave been primarily attributed to the formation ofDCVC, whose metabolites can rearrange to formpotent acylating species. Subsequent acylation ofproteins and DNA may lead to cytotoxicity andmutagenesis (Anders et al., 1988; Goeptar et al.,1995).
The toxic effects of TRI are believed to be aresult of repeated exposures to TRI over an ex-
tended period of time. This is true of severalchemicals, and chemical-induced injury to notonly the kidney, but to many other organs, isoften the result of repeated or chronic exposure toa chemical over a longer period of time. Becauseof this, efforts have been made to develop modelsthat can be used for the study of long-term chem-ical-induced injury. An in vitro model that mimicsthe in vivo state would greatly facilitate the studyof chemical-induced renal injury. A model hasbeen previously developed using primary culturesof proximal tubular (PT) and distal tubular (DT)cells from rat kidney (Lash et al., 1995). Thesecultured cells maintain similar biochemical func-tion as the freshly isolated cells. For example,levels of marker enzymes, such as GGT and hex-okinase (EC 2.7.1.1), in cultured PT and DT cellsand the activities of alkaline phosphatase, cellularenergy metabolism enzymes, and the expression ofcytokeratins, are maintained over a period of atleast 5 days. Furthermore, the same cell-specificpatterns of susceptibility to chemical toxicantssuch as methyl vinyl ketone and tert-butyl hy-droperoxide, monitored with freshly isolated cells,were also observed in cultures of PT and DT cells.
The goal of the work presented in this study isto use these previously validated cultures to studythe cytotoxicity of TRI and DCVC over an ex-tended period of time (days vs. hours). Previouswork showed that both TRI and DCVC causecytotoxicity in freshly isolated PT and DT cells,but these studies only examined the effects ofthese chemicals after 2-h incubations (Lash et al.,1994; Cummings et al., 2000). Although the PTcells are the major in vivo target cell populationfor TRI and DCVC, examination of cytotoxicityin a well-characterized non-target cell population(i.e. DT cells) will provide additional insight intofactors that are responsible for the toxicity. It hasalso been demonstrated that freshly isolated PTand DT cells express CYP2E1, CYP2B1/2,CYP2C11, and CYP4A2/3 but not CYP3A1/2(Cummings et al., 1999). The expression of theseenzymes in primary cultures of PT and DT cells,however, has not been determined. Examinationof the cytotoxicity of TRI and its metaboliteDCVC in these cultures will advance the knowl-edge of the mechanism(s) by which these agents
B.S. Cummings et al. / Toxicology 150 (2000) 83–98 85
cause cellular injury. Preliminary results of thiswork have been presented.3
2. Materials and methods
2.1. Materials
Percoll, collagenase (Type I; EC 3.4.24.3), pow-dered 1:1 mixture of Dulbecco’s modified Eagle’smedium/Ham’s F12 (DMEM/F12), HEPES,bovine serum albumin (BSA; fraction V), g-glu-tamyl-p-nitroanilide, p-nitrophenylphosphate,penicillin G, streptomycin sulfate, amphotericinB, insulin (from bovine pancreas), human trans-ferrin, sodium selenite, hydrocortisone, 3,3%,5-tri-iodo-DL-thyronine, thyrocalcitonin (from bovinethyroid gland), and ciprofibrate were purchasedfrom Sigma (St. Louis, MO). Epidermal growthfactor was purchased from Upstate Biotechnology(Lake Placid, NY). Polystyrene tissue culturedishes were purchased from Falcon or Corningand Teflon cell scrappers were purchased fromFalcon. Rat CYP4A1 cDNA, which recognizesmRNA for several CYP4A isoforms, and mouse7S cDNA were generously provided by Dr FrankGonzalez (National Cancer Institute, Bethesda,MD) and Dr Allan Balmain (Beatson Institute forCancer Research, Glasgow, UK), respectively.Antibodies for rat CYP2E1, CYP2C11, andCYP3A1/2 were purchased from Oxford Biomedi-cal (Rochester Hills, MI). Antibody for ratCYP2B1/2 was purchased from Xenotech (Kan-sas City, KS). Antibody for rat CYP4A, whichrecognizes multiple CYP4A isoforms, was pur-chased from Gentest (Woburn, MA).
2.2. Isolation of rat renal PT and DT cells
Isolated renal cortical cells were obtained bycollagenase perfusion (Jones et al., 1979) frommale Fischer 344 rats (150–250 g; Charles RiverLaboratories, Wilmington, MA). To obtain en-
riched populations of renal PT and DT cells,cortical cells were subjected to density-gradientcentrifugation in Percoll as described previously(Lash and Tokarz, 1989). Briefly, after anesthesiawith pentobarbital (50 mg/ml; 0.11 ml/100 g bodyweight), rats were injected with 0.3 ml of 0.2%(w/v) heparin in 0.9% (w/v) saline. The aortabelow the renal arteries was then cannulated witha 19-gauge steel cannula and the kidneys weresubjected to initial recirculating perfusion at 8ml/min with a calcium-free, EGTA-containingHanks’ Buffer (Hanks’ I, supplemented with 25mM NaHCO3, 25 mM HEPES, pH 7.4, 0.5 mMEGTA, and 0.2% (w/v) BSA). All buffers werecontinuously bubbled with 95% O2/5% CO2 andwere maintained at 37°C. The kidneys were thenperfused with Hanks’ buffer supplemented with 4mM CaCl2 and collagenase (0.1%, w/v), in arecirculating manner at 5 ml/min for 13–18 min.Cells were dispersed by mechanical disruption.The cortical cells were collected by low-speedcentrifugation (100×g for 30 s) and subjected todensity-gradient centrifugation in Percoll (20%,v/v). Activities of marker enzymes and functionalassays were used to confirm the identity andpurity of the two cell populations (Lash andTokarz, 1989). Cell concentrations were deter-mined in the presence of 0.2% (w/v) trypan blue ina hemacytometer, and cell viability was estimatedby measuring the fraction of cells that excludedtrypan blue.
2.3. Primary culture of PT and DT cells
Primary cultures of PT and DT cells were pre-pared as described previously (Lash et al., 1995).Isolation of PT and DT cells was achieved asexplained above and cells were resuspended in 2ml of Krebs–Henseleit Buffer (118 mM NaCl, 4.8mM KCl, 0.96 mM KH2PO4, 0.12 mMMgSO4 · 7H2O, 25 mM NaHCO3) containing 25mM HEPES, and diluted to 30 ml with cellculture media. Basal media was a 1:1 mixture ofDMEM:F12. Standard supplementation for bothPT and DT cells included 15 mM HEPES, pH7.4, 20 mM NaHCO3, antibiotics for day 0through day 3 only (192 IU penicillin G/ml+200mg streptomycin sulfate/ml) to inhibit bacterial
3 Cummings, B.S., Lash, L.H., 1998. Toxicity andmetabolism of trichloroethylene and S-(1,2-dichlorovinyl)-L-cysteine in freshly isolated and cultured proximal tubular anddistal tubular cells from rat kidney. Toxicol. Sci. 42 (1-S), 380.
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growth, 2.5 mg amphotericin B/ml to inhibit fungalgrowth, 5 mg bovine insulin/ml, 5 mg human trans-ferrin/ml, 30 nM sodium selenite, 100 ng hydrocor-tisone/ml, and 100 ng epidermal growth factor/ml.PT cells also received 7.5 pg 3,3%,5-triiodo-DL-thy-ronine/ml, while DT cells received 5 ng thyrocalci-tonin/ml. Cells were seeded at densities of 50–100mg protein per cm2 (0.5–1.0×106 cells/ml) inpolystyrene culture dishes. Cultures were grown at37°C in a humidified incubator under an atmo-sphere of 95% air/5% CO2 at pH 7.4. Cultures wereallowed to attach and grow for at least 24 h priorto treatment with any agent. Cells were harvestedfrom the dishes by either scraping the plates witha Teflon scraper or by brief incubation with 0.05%(w/v) trypsin (EC 3.4.21.4)/0.2% (w/v) EDTA inCa2+- and Mg2+-free Hanks’ buffer.
2.4. Assay of cytotoxicity
Primary cultures of PT and DT cells were incu-bated with either medium or medium containingthe indicated concentrations of TRI or DCVC foreither short-term exposures (1 or 3 h) or long-termexposures (24 or 72 h). Cell viability at the conclu-sion of these incubations was estimated by deter-mining the release of lactate dehydrogenase (LDH)from cells after various incubations and at varioustimes (Lash et al., 1995). For cytotoxicity assays inprimary cultures of PT and DT cells, LDH releasefrom cells was measured by determining LDHactivity (measured spectrophotometrically asNADH oxidation at 340 nm) in media and, afterremoval of media, washing cells with phosphate-buffered saline (PBS) and solubilization of cellswith 0.1% (v/v) Triton X-100, in total cells. Thefraction of LDH release was an index of irre-versible injury:
%LDH release=LDH activity in media/(LDH activity in media+LDH activity in total cells)×100%.
2.5. DNA and enzyme assays
DNA content was measured as the relativefluorescence of the DNA-diamidinophenylindole
complex with 360-nm excitation and 450-nm emis-sion, according to Sorger and Germinario (1983),with double-stranded, calf thymus DNA as astandard. GGT activity was measured at 410 nmby monitoring p-nitroanilide formation (o=8800M−1 cm−1) with g-glutamyl-p-nitroanilide andglycylglycine as substrates according to Orlowskiand Meister (1963). GST (EC 2.5.1.18) activitywas determined by measuring the formation of2,4-dinitrophenylglutathione spectrophotometri-cally at 340 nm (o=9600 M−1 cm−1) with 1 mM1-chloro-2,4-dinitrobenzene and 1 mM GSH assubstrates, as described by Habig (1974). Glu-tathione disulfide (GSSG) reductase (GRD) (EC1.6.4.2) activity was measured spectrophotometri-cally by observing the decrease in NADPH (0.1mM) absorbance at 340 nm (o=9600 M−1 cm−1)in the presence of GSSG (Eklow et al., 1984).GSH peroxidase (GPX) (EC 1.11.1.9) activity wasmeasured spectrophotometrically by recording thechange in absorbance of NADPH (0.2 mM) at340 nm (o=6220 M−1 cm−1) in the presence ofGRD (1 U/ml), NaN3 (1 mM), GSH (1 mM), andH2O2 (0.25 mM), as described by Lawrence andBurk (1976). g-Glutamylcysteine synthetase(GCS) (EC 6.3.2.2) activity was determined bymeasuring the decrease in absorbance of NADH(o=6220 M−1 cm−1) after oxidation by pyruvatethat is generated by the formation of ADP fromL-glutamate (10 mM) and L-a-aminobutyrate (10mM) in the presence of Mg-ATP (20 mM) (Seeligand Meister, 1984). Hexokinase activity was de-termined by measuring the increase in NADPHabsorbance at 340 nm (o=6220 M−1 cm−1) ascatalyzed by glucose-6-phosphate dehydrogenase(EC 1.1.1.49) (2 U/ml) in the presence of ATP (50mM) and glucose (15 mM) (Joshi and Jagan-nathan, 1966). Protein determination was doneusing the bicionchoninic acid (BCA) protein de-termination kit from Sigma, using BSA as astandard.
2.6. Northern blot analyses
Total RNA was isolated from rat liver andkidney homogenates or PT and DT cells by acidphenol extraction using the TRIZOL® extractionkit (Gibco BRL, Gaithersburg, MD). Blots were
B.S. Cummings et al. / Toxicology 150 (2000) 83–98 87
then incubated with probes to CYP2E1, CYP3A1,and CYP4A1 mRNA as described previously(Zangar et al., 1995). Total RNA (10 mg/lane) wasfractionated on a formaldehyde/agarose gel,transferred to a nylon membrane, probed withcDNAs complementary to the indicated mRNA,or stripped and reprobed with a cDNA to mouse7S RNA to standardize mRNA loading, autora-diographed, and band density was determined byscanning laser densitometry.
2.7. Western blot analyses of indi6idual P450isoforms
The expression of individual P450 isoforms inmicrosomes from the cultured cells was analyzedby subjecting microsomes to SDS-PAGE on a 7or 10% gel followed by transfer of the fraction-ated protein to nitrocellulose. Nitrocellulose mem-branes were then incubated with the indicatedantibodies. Alkaline phosphatase-conjugated sec-ondary enzymes and substrate were used to detectprotein bands. Band densities were determined byscanning laser densitometry. For CYP4A detec-tion in rats, the method of Okita et al. (1998) wasused to achieve separation of CYP4A into itsindividual isoforms. This method involves subject-ing small amounts of microsomes to SDS-PAGEafter pre-running the gel for 4 h at 25 mA. Afterloading, samples were allowed to migrate until the40 kDa marker band was approximately 10% (10mm) from the bottom of the gel. The gel was thentransferred to a nitrocellulose membrane and pro-cessed as described above.
2.8. Immunohistochemical staining forcytokeratins and 6imentins
Cultures of PT and DT cells were grown on35-mm polystyrene dishes. Cytokeratins weremonitored as an epithelial cell marker by indirectimmunofluorescent staining as described by Cho-pra et al. (1987). Vimentins were monitored as anendothelial cell marker and should not be presentin control cells (Vamvakas et al., 1990). Afterfixation with 3.7% (v/v) formalin in PBS, cellswere washed several times with PBS containingsaponin (0.1% w/v), then incubated with a-keratin
conjugated to FITC antibody from guinea pig ora mouse anti-donkey vimentin antibody (Sigma).After 1 h, cultures were washed with PBS andviewed with a Zeiss Triple-Laser Scanning Confo-cal Microscope (LSM) 310 with integrated work-station at the Confocal Imaging Core Facility inthe School of Medicine at Wayne State University(Detroit, MI). For vimentin, cultures were incu-bated with a secondary antibody solution conju-gated to Texas Red.
2.9. Data analysis
All values are means9S.D. of measurementsmade on the indicated number of separate prepa-rations. Significant differences between means fordata were first assessed by a one-way analysis ofvariance. When significant F values were ob-tained, the Fisher’s protected least significance ttest was performed to determine which meanswere significantly different from one another, withtwo-tail probabilitiesB0.05 consideredsignificant.
3. Results
3.1. Acti6ity of GSH-dependent enzymes inprimary cultures of PT and DT cells
Freshly isolated PT and DT cells were placed inprimary culture and reached confluency in 5–7days (Fig. 1). Cells formed a monolayer andexhibited epithelial morphology, as demonstratedpreviously (Lash et al., 1995). Activities of GGT,GRD, GPX, GST, GCS, and hexokinase weremeasured in freshly isolated PT and DT cells(Day 0= freshly isolated cells), and in cells from 3and 5-day-old cultures. For the most part, theactivity of all these enzymes did not change sig-nificantly over the time period tested with theexception of GCS, which decreased initially in PTcells, but subsequently recovered to levels foundin freshly isolated cells (Fig. 2). In the rat kidney,GGT is a marker enzyme for PT cells, whilehexokinase is a marker enzyme for DT cells (Lashand Tokarz, 1989). The activity of GGT in freshlyisolated PT cells is typically 5–10-fold higher than
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in DT cells, whereas that of hexokinase is 5–10-fold higher in freshly isolated DT cells relative toPT cells. Primary cultures of PT and DT cellsmaintained these expected ratios of GGT andhexokinase activities, even after 5 days in culture.
3.2. Expression of P450 isoforms in primarycultures of PT and DT cells
The P450 isoforms studied were those shownpreviously to be expressed in freshly isolated PTand DT cells (Cummings et al., 1999). Of all theP450 isoforms studied, and despite the use ofinducers, the levels of CYP2E1, CYP2C11,CYP2B1/2, or CYP3A1/2 were below the limit ofdetection by either northern or western blot anal-ysis (data not shown). Northern blot analysis,using a cDNA probe to CYP4A1 mRNA thatrecognizes all CYP4A mRNA family members,detected CYP4A mRNA in primary cultures ofDT cells, but not PT cells, after 5 days of culture(Fig. 3A). Treatment of cultures for 3 days withthe peroxisomal proliferator ciprofibrate (100 mM)increased CYP4A mRNA expression in DT cells(Fig. 3A). Differences in CYP4A mRNA expres-sion were not associated with differences in load-ing as determined by the expression of 7S RNA(Fig. 3B). CYP4A mRNA was still undetectablein PT cells, even after treatment with ciprofibrate.Western blot analysis using a polyclonal antibodyto rat CYP4A showed a detectable level ofCYP4A was expressed in microsomes preparedfrom primary cultures of DT cells (Fig. 4, lane 1)and a faint band that coeluted with CYP4A wasalso detected in primary cultures of PT cells (Fig.4, lane 2). The presence of a small amount ofimmunoreactive protein and the absence of de-tectable CYP4A mRNA in cultures of PT cells,suggest that either CYP4A mRNA is present butis below the limit of detection or that transcrip-tion had stopped before translation in those cells.
3.3. Toxicity of TRI and DCVC in primarycultures of PT and DT cells after short-termexposure
As primary cultures of PT and DT cells ap-peared to maintain activities of GSH-dependentenzymes, and as the expression of at least oneP450 isoform was maintained, the toxicity of TRIand DCVC in primary cultures of PT and DTcells was studied. The short-term (0–6 h) toxicityof TRI (0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM) inprimary cultures of PT and DT cells was studied
Fig. 1. Photomicrograph of primary cultures of proximaltubular (PT) and distal tubular (DT) cells after 5 days of cellgrowth. Freshly isolated PT (A) and DT (B) cells were seededonto a 35-mm polystyrene dish at a density of 0.5–1.0×106
cells/ml. Cells were grown for 5 days and photomicrographswere taken at 100× magnification on a Carl-Zeiss ConfocalLaser Microscope. Bar=5 mm.
B.S. Cummings et al. / Toxicology 150 (2000) 83–98 89
Fig. 2. Activity of glutathione (GSH)-dependent enzymes and hexokinase in 0, 3, and 5 day old primary cultures of proximal tubular(PT) and distal tubular (DT) cells. Freshly isolated PT and DT cells were seeded at a density of 0.5–1.0×106 cells/ml and allowedto grow for 3 or 5 days. At each time point, cells were isolated and the activities of GGT, GRD, GPX, GST, GCS, and hexokinasewere determined and compared to activities in freshly isolated cells (Day 0). Data represent the mean9S.D. of at least threeseparate cell isolations.
by assessment of LDH release (Fig. 5A,B). TRIwas not cytotoxic at any concentration or timepoint tested.
In contrast to the results with TRI, DCVC (0,0.1, 0.2, 0.5, and 1 mM) was highly cytotoxic inprimary cultures of both PT and DT cells (Fig. 6).The sensitivity of the PT cells to DCVC wasevidenced by the significant increase in LDH re-lease after 1 h of exposure to 0.2 mM DCVC orafter 2 h exposure to 0.1 mM DCVC (Fig. 6A).At the 1- and 2-h time points, primary cultures ofDT cells were less susceptible to DCVC-inducedcytotoxicity than PT cells, as evidenced by theabsence of a significant increase in LDH release atDCVC concentrations below 0.5 mM (Fig. 6B).After 3-h incubations, the extent of LDH releaseinduced by DCVC was similar to or modestlyhigher in DT cells than in PT cells.
3.4. Toxicity of TRI and DCVC in primarycultures of PT and DT cells after long-termexposures
The toxicity of TRI (0, 0.1, 0.2, 0.5, 1, 2, 5, and10 mM) in primary cultures of PT and DT cellsfollowing 24 or 72 h of treatment was assessed bymeasuring the %LDH released from these cul-tures. TRI was only mildly cytotoxic to primarycultures of both PT and DT cells after 72 h ofexposure to high concentrations of TRI (5 and 10
mM) (Fig. 7A,B). The cytotoxicity of TRI wasalso time-dependent. In contrast, DCVC washighly cytotoxic in primary cultures of both PTand DT cells, causing significant increases inLDH release at concentrations as low as 0.5 and0.2 mM after just 24 h in PT and DT cells,respectively (Fig. 8A,B). DCVC cytotoxicity wastime- and concentration-dependent in primarycultures of PT cells. In primary cultures of DTcells, DCVC cytotoxicity was time and concentra-tion-dependent up to 5 mM. LDH release wasmarkedly lower in DT cells incubated for 72 hwith 7.5 or 10 mM DCVC because the totalamount of LDH activity was markedly decreased(data not shown), consistent with a loss of cellsand degradation of the released enzyme. Whereasthe acute cytotoxicity data (cf. Fig. 6) suggestedcomparable sensitivity of the two cell populationsto DCVC, the data from incubations for 72 hsuggest higher sensitivity of the DT cells afterprolonged (i.e. 72 h) incubations.
3.5. Effect of TRI and DCVC on cellular proteinand DNA le6els
No effect was observed on either cellularprotein or DNA levels after 72 h of treatmentwith 0, 0.1, 0.2, 0.5, 1, 2, 5, or 10 mM concentra-tions of TRI (data not shown). In contrast,DCVC significantly decreased cellular protein and
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DNA levels after 72 h of treatment (Fig. 9A,B):concentrations greater than 1 mM decreased cel-lular protein significantly and concentrationsgreater than 2 mM resulted in a maximal decreasein cellular protein content of \60% in both cellcultures. Treatment with relatively low concentra-tions of DCVC (0.1 mM) actually resulted in aslight, but significant, increase in cellular DNAlevels in DT cells, but not in PT cells, whileconcentrations greater than 2 mM resulted insignificant decreases in PT cells (Fig. 9B). Theincrease in DNA content in DT cells suggests thepossibility that increased cellular mitosis was ini-tiated by the treatment with low concentrations ofDCVC.
3.6. Effect of TRI and DCVC on cytokeratin and6imentin expression
To assess the effects of TRI (10 mM) andDCVC (10 mM) treatment on maintenance ofepithelial phenotype and on the possible inductionof mitosis and cellular regeneration, expression ofcytokeratin and vimentin in primary cultures ofPT and DT cells was determined by immunohisto-chemical staining. The dose of DCVC was chosenso that there would be no cytotoxicity over the72-h incubation period. A significant increase wasobserved in LDH release with 20 mM DCVC butnot with 10 mM DCVC (data not shown). Freshlyisolated PT and DT cells were seeded, allowed 24h to attach, and then were treated with either TRIor DCVC for 24 or 72 h. After this time, mediacontaining TRI or DCVC were removed and thecells were allowed to recover for 0, 3, 6, or 24 h inthe appropriate culture medium lacking TRI orDCVC. After the recovery time period, cells werewashed and stained with the appropriate anti-body. At no time did TRI or DCVC appear toalter cytokeratin staining. Alterations in vimentinexpression were only detected after 72 h of expo-sure and 3 h of recovery. At this time point,treatment of primary cultures of either PT or DTcells with TRI or DCVC resulted in no apparentdecreases in cytokeratin staining (Figs. 10 and12). Treatment of primary cultures with TRI in-creased vimentin expression in both cell cultures,but more prominently in DT cells (Fig. 11).
Fig. 3. Effect of ciprofibrate on expression of CYP4A mRNAin primary cultures of proximal tubular (PT) and distal tubu-lar (DT) cells. Freshly isolated PT and DT cells were seeded ata density of 0.5–1.0×106 cells/ml and were allowed to growfor 24 h. After 24 h, the media were removed and replacedwith media containing solvent control ([ethanol]B1.0%, v/v)or 100 mM ciprofibrate. Cultures were then allowed to growfor an additional 72 h. Total RNA (10 mg/lane) was fraction-ated on a formaldehyde/agarose gel, transferred to a nylonmembrane, probed with a cDNA complementary to ratCYP4A1 or mouse 7S RNA (loading standard) and autoradio-graphed.
Fig. 4. Western blot analysis of CYP4A expression in micro-somes isolated from primary cultures of proximal tubular (PT)and distal tubular (DT) cells. Freshly isolated PT and DT cellswere seeded at a density of 0.5–1.0×106 cells/ml. Cells wereallowed to grow for 4 days, after which cells were isolated andmicrosomes were prepared. Microsomes (60 mg) were subjectedto SDS-PAGE followed by transfer to a nitrocellulose mem-brane that was then incubated with a polyclonal goat anti-ratCYP4A antibody solution.
B.S. Cummings et al. / Toxicology 150 (2000) 83–98 91
Fig. 5. Toxicity of trichloroethylene (TRI) in primary cultures of proximal tubular (PT) and distal tubular (DT) cells after short-termexposure. Freshly isolated PT and DT cells were seeded at a density of 0.5–1.0×106 cells/ml and allowed to grow to confluency(approximately 4–5 days). The toxicity of TRI at the indicated time points was then determined by assessment of lactatedehydrogenase (LDH) release from the cells. The concentrations of TRI tested (X-axis) are presented on a log-scale · %LDHrelease= (LDH activity in media)/(LDH activity in media+LDH activity in cells)×100%. LDH activity was determined by thedecrease in NADH absorbance at 340 nm. Data are the mean9S.D. of at least three separate measurements.
Fig. 6. Toxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in primary cultures of proximal tubular (PT) and distal tubular (DT)cells after short-term exposure. Freshly isolated PT and DT cells were seeded at a density of 0.5–1.0×106 cells/ml and allowed togrow to confluency (approximately 4–5 days). The toxicity of DCVC at the indicated time points was then determined by assessmentof lactate dehydrogenase (LDH) release, as described in the legend to Fig. 5. Data are the mean9S.D. of at least three separatemeasurements. * Significant difference (PB0.05) from control.
DCVC slightly increased vimentin expression incultures of both PT and DT cells (Fig. 13). Im-portantly, vimentin was not expressed in controlcells, as expected for normal epithelial cells.
4. Discussion
This investigation describes for the first time,studies in primary cultures of PT and DT cellsfrom rat kidney of both acute and longer-term
incubations with TRI and its metabolite DCVCand data on activity and expression of severaldrug-metabolizing enzymes. Data from this studyshowed that primary cultures of PT and DT cellsare capable of maintaining most of their activitiesof GSH-dependent enzymes and hexokinase, thusmimicking closely freshly isolated cells. More im-portantly, the ratio of GGT to hexokinase inthese cultures was maintained as compared withthat in freshly isolated PT and DT cells (cf. Fig.2). Loss of GGT activity is a sensitive indicator of
B.S. Cummings et al. / Toxicology 150 (2000) 83–9892
dedifferentiation in a renal PT cell culture. Themaintenance of GST activity with CDNB as sub-strate gives only a general indication of levels ofGST and does not provide any information onspecific isoforms. However, it was recently re-ported that GSTa is the primary class of GSTisoforms expressed in freshly isolated PT and DTcells from the rat (Cummings et al., 2000). Hence,
the activity data reported here likely reflects activ-ity primarily due to GSTa, although one cannotexclude the possibility that expression of anotherGST isoform increased during culture. Addition-ally, primary cultures of PT and DT cells incu-bated with media only (i.e. controls) maintainedexpression of cytokeratin and did not expressvimentin (cf. Figs. 10–13). Thus, these cells ap-
Fig. 7. Toxicity of trichloroethylene (TRI) in primary cultures of proximal tubular (PT) and distal tubular (DT) cells after longterm-exposure. Freshly isolated PT and DT cells were seeded at a density of 0.5–1.0×106 cells/ml and allowed to grow toconfluency (approximately 4–5 days). The toxicity of TRI at the indicated time points was then determined by assessment of lactatedehydrogenase (LDH) release, as described in the legend to Fig. 5. The concentrations of TRI tested (X-axis) are presented on alog-scale. Data are the mean9S.D. of at least three separate measurements. Control values for %LDH release in PT cells at 1 and3 days were 20.1 and 22.9%, respectively. Control values for %LDH release in DT cells at 1 and 3 days are 19.2 and 25.9%,respectively. * Significant difference (PB0.05) from control.
Fig. 8. Toxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in primary cultures of proximal tubular (PT) and distal tubular (DT)cells after long-term exposure. Freshly isolated PT and DT cells were seeded at a density of 0.5–1.0×106 cells/ml and allowed togrow to confluency (approximately 4–5 days). The toxicity of DCVC at the indicated time points was then determined by assessmentof lactate dehydrogenase (LDH) release, as described in the legend to Fig. 5. Data are the mean9S.D. of at least three separatemeasurements. The concentrations of DCVC tested (X-axis) are presented on a log-scale. Control values for %LDH release in PTcells at Day-1 and Day-3 were 11.091.1 and 20.494.1, respectively. Control values for %LDH release in DT cells at Day-1 andDay-3 were 12.291.6 and 21.292.6, respectively. * Significant difference (PB0.05) from control.
B.S. Cummings et al. / Toxicology 150 (2000) 83–98 93
Fig. 9. Effect of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) on cellular protein and DNA levels in primary cultures of proximal tubular(PT) and distal tubular (DT) cells. Freshly isolated PT and DT cells were seeded at a density of 0.5–1.0×106 cells/ml and allowed24 h to attach, at which time DCVC at the indicated concentrations was added to the media. Cells were then allowed to grow for72 h. Cellular protein and DNA levels were determined as described in Section 2. Results are the mean9S.D. of at least threeseparate experiments. Concentrations of DCVC tested (X-axis) are presented on a log-scale. Control values for protein levels in PTand DT cells were 0.14690.005 and 0.14190.014 mg/ml, respectively. Control values for DNA levels in PT and DT cells were22.592.9 and 15.991.8 mg/ml, respectively. * Significant difference (PB0.05) from control.
pear to be epithelial in nature and to be validmodels in which to study chemical-inducednephrotoxicity involving GSH-dependent bioacti-vation enzymes.
With the exception of CYP4A, the expressionof all P450 isoforms tested was below the level ofdetection in primary cultures of PT and DT cells.Loss of P450 expression in primary cultures is notuncommon, as many studies have reported such aphenomenon (Kocarek et al., 1993; Zangar et al.,1995). The maintenance of CYP4A isoform ex-pression over that of others (viz., CYP2C,CYP2B, CYP2E1) may be due to its higher levelsof expression in freshly isolated cells as comparedto that of the other isoforms (Cummings et al.,1999). The loss of CYP4A over time is likely theresult of decreased transcription, as CYP4A wasinducible with ciprofibrate (cf. Fig. 3). This, com-bined with the inability to detect the mRNA foreither CYP2E1 or CYP2B, suggest that a decreasein transcription causes the decrease in expressionof these P450 isoforms. Although the method ofOkita et al. (1998) was previously used to measureexpression of CYP4A isoforms and detected bothCYP4A2 and CYP4A3 in freshly isolated rat kid-ney cells (Cummings et al., 1999), only a singleband was observed in the DT cell primary cul-tures. The occurrence of only a single band may
represent either an inability to separate multipleisoforms or may indicate that only one of theCYP4A isoenzymes is expressed in culture. It isbelieved that the latter case is true, and we suggestthat the single band is probably CYP4A2, becauseit was expressed at higher levels than CYP4A3 infreshly isolated cells. However, this requires fur-ther study to be confirmed.
TRI cytotoxicity after short-term treatment (B6 h) of primary cultures of PT and DT cells wasless than that measured in freshly isolated PT andDT cells (Cummings et al., 2000 and Figs. 5 and6). Primary cultures of PT and DT cells areapproximately 70–90% confluent at the start ofthe cytotoxicity experiments and typically havelow (B10%) basal levels of LDH release (Lash etal., 1995). In contrast, freshly isolated PT and DTcells are in suspension and have moderatelyhigher basal levels of LDH release than primarycultures (i.e. 10–20%) (Lash and Tokarz, 1989;Lash et al., 1994; Cummings et al., 2000). Thismay partially account for the difference in theextent of cytotoxicity observed in the two in vitromodels. However, the alterations in expression ofsome enzymes may play a role inasmuch asmetabolism is required for toxicity. It should benoted that TRI-induced cytotoxicity in freshlyisolated PT and DT cells is still only modest, with
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Fig. 10. Effect of trichloroethylene (TRI) on cytokeratin expression in primary cultures of proximal tubular (PT) and distal tubular(DT) cells. Freshly isolated PT (A) and DT (B) cells were seeded at a density of 0.5–1.0×106 cells/ml and allowed to grow for 24h prior to treatment with TRI (10 mM). After treatment for 72 h, cells were washed twice with sterile PBS and allowed to recoverin appropriate media for 3 h. Cytokeratin expression was visualized using a monoclonal FITC-conjugated anti-mouse cytokeratinantibody. Photomicrographs were taken at 100× magnification on a Zeiss Confocal Laser Microscope. Bar=5 mm.Fig. 11. Effect of trichloroethylene (TRI) on vimentin expression in primary cultures of proximal tubular (PT) and distal tubular(DT) cells. Freshly isolated PT (A) and DT (B) cells were seeded at a density of 0.5–1.0×106 cells/ml and allowed to grow for 24h prior to treatment with TRI (10 mM). After treatment for 72 h, cells were washed twice with sterile PBS and allowed to recoverin appropriate media for 3 h. Vimentin expression was visualized using a monoclonal Texas Red-conjugated anti-mouse vimentinantibody. Photomicrographs were taken at 100× magnification on a Zeiss Confocal Laser Microscope. Bar=5 mm.
B.S. Cummings et al. / Toxicology 150 (2000) 83–98 95
Fig. 12. Effect of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) on cytokeratin expression in primary cultures of proximal tubular (PT)and distal tubular (DT) cells. Freshly isolated PT (A) and DT (B) cells were seeded at a density of 0.5–1.0×106 cells/ml and allowedto grow for 24 h prior to treatment with DCVC (10 mM). After treatment for 72 h, cells were washed twice with sterile PBS andallowed to recover in appropriate media for 3 h. Cytokeratin expression was visualized using a monoclonal FITC-conjugatedanti-mouse cytokeratin antibody. Photomicrographs were taken at 100× magnification on a Zeiss Confocal Laser Microscope.Bar=5 mm.Fig. 13. Effect of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) on vimentin expression in primary cultures of proximal tubular (PT) anddistal tubular (DT) cells. Freshly isolated PT (A) and DT (B) cells were seeded at a density of 0.5–1.0×106 cells/ml and allowedto grow for 24 h prior to treatment with DCVC (10 mM). After treatment for 72 h, cells were washed twice with sterile PBS andallowed to recover in appropriate media for 3 h. Vimentin expression was visualized using a monoclonal Texas Red-conjugatedanti-mouse vimentin antibody. Photomicrographs were taken at 100× magnification on a Zeiss Confocal Laser Microscope.Bar=5 mm.
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an �20% increase in LDH release. Treatment ofprimary cultures of PT and DT cells with TRI forlonger time periods (\24 h) resulted in a higheramount of cytotoxicity, as assessed by LDH re-lease. TRI was cytotoxic after 3 days of treatmentof cells with concentrations of at least 5 mM TRI(cf. Fig. 7). The two cell populations appeared tobe equally susceptible.
Unlike incubations with TRI, incubations withDCVC caused marked and significant increases inLDH release in primary cultures of PT and DTcells after both long-term and short-term treat-ments. The lowest concentration of DCVC testedthat caused cytotoxicity after only 1 h of incuba-tion was 0.2 mM in PT cells and 0.5 mM in DTcells (cf. Fig. 6). The lowest concentration ofDCVC tested (i.e. 0.1 mM) caused cytotoxicityafter 2 h in PT cells and after 3 h in DT cells.Thus, at 1- and 2-h incubations, PT cells exhibitedgreater increases in LDH release than DT cells atDCVC concentrations below 0.5 mM. At thehighest concentration of DCVC and longest incu-bation time tested in the acute cytotoxicity study(i.e. 1 mM and 3 h), PT cells exhibited less LDHrelease than DT cells (�60 vs. �100%). How-ever, this was not due to diminished toxicity butto a decrease in total LDH activity as a conse-quence of cell death and degradation of releasedLDH (data not shown). Hence, one cannot con-clude from the acute cytotoxicity data that eithercell population exhibited a significantly differentsusceptibility to DCVC, even though the PT cellsare the in vivo target cell population.
In the long-term cytotoxicity experiments, TRIcaused the same amount of LDH release in bothcell populations after 24 and 72 h incubations.Similarly, PT and DT cells incubated with DCVCfor 24 h caused the same amount of LDH releasein the two cell populations. However, PT cellsincubated with DCVC for 72 h exhibited a maxi-mal amount of LDH release of �80% at DCVCconcentrations of ]2 mM. In contrast, DT cellsincubated with DCVC concentrations of 5 or 10mM for 72 h exhibited markedly less LDH releasethan either DT cells incubated with 52 mMDCVC or PT cells incubated with 5 or 10 mMDCVC. Again, these apparent decreases in cyto-toxicity are likely due to cell death and degrada-
tion of released LDH, suggesting that the DT cellsare actually more susceptible to irreversible injurythan PT cells at these higher concentrations ofDCVC.
The observation that DT cells are equally sus-ceptible, and under certain conditions, more sus-ceptible to injury from DCVC than PT cells,suggest that a major determinant in the in vivospecificity of DCVC-induced cellular injury doesnot involve significant differences in intracellularmetabolism or availability of intracellular targetsfor reactive metabolites. Rather, it is likely thatthe PT cells are the primary cell type that becomesinjured after exposure to DCVC because it is thefirst one to be exposed to it and the presence ofhighly active transporters on the PT cell plasmamembranes delivers DCVC to bioactivation en-zymes inside the cell. Although there are somedifferences in bioactivation enzymes for DCVC inPT and DT cells (Lash et al., 1994), both celltypes would generate the same reactive metaboliteand both cell types possess a high density ofmitochondria. The latter suggests that similar in-tracellular targets are present, and that the mecha-nism of action is likely to be the same in both celltypes.
The slight, but significant increase in DNAlevels in DT cells treated with a relatively lowconcentration of DCVC (0.1 mM), suggests thatsublethal injury and cellular regeneration mayoccur in these cultures. DCVC can stimulate cel-lular proliferation under appropriate conditions(Hatzinger et al., 1988; Vamvakas et al., 1989;Eyre et al., 1995a,b; Kays and Schnellmann,1995). Cytokeratin and vimentin expression weremeasured as markers for these processes in pri-mary cultures of PT and DT cells. (cf. Figs.10–13). Cytokeratins are expressed in differenti-ated epithelial cells. In contrast, new cell growthin a proliferating cell population is comprised ofcells that may be considered dedifferentiated be-cause they do not express the phenotype of themature epithelial cells. Vimentin can be used as amarker for sublethal cellular injury and regenera-tion (Hatzinger et al., 1988) as it is normallyexpressed in endothelial, but not epithelial, cells.Exposure of primary cultures of PT and DT cellsto TRI (10 mM) or DCVC (10 mM) for 3 days
B.S. Cummings et al. / Toxicology 150 (2000) 83–98 97
followed by 3 h of recovery resulted in no appar-ent alteration in cytokeratin staining, suggestingthat under the present incubation conditions, nei-ther chemical causes significant loss of this char-acteristic in the two cell populations. Undernormal culture conditions, PT and DT cells donot express vimentin (cf. Figs. 11 and 13). Expo-sure of primary cultures of PT and DT cells toTRI, however, resulted in the detection of lowlevels of vimentin primarily in DT cells, whileDCVC treatment resulted in increased expressionof low levels of vimentin in both PT and DT cells.These data are consistent with the suggestion thatboth TRI and DCVC are causing sublethal injuryand cellular proliferation. The mechanisms under-lying the increase in vimentin expression in thesecells are unknown. The absence of any apparentchange in cytokeratin levels but the evident in-crease in vimentin are not inconsistent observa-tions. Rather, this is likely an issue of sensitivity,in that with cytokeratins, it is difficult to observea small decrease from high baseline levels whereaswith vimentin, an increase from a baseline of zeroexpression was observed.
In summary, PT and DT cells in confluentprimary culture appear to maintain their epithelialmorphology and expression of several GSH-de-pendent enzymes. Thus, these cells should proveuseful as models for the study of chemical-in-duced injury to the kidney where the bioactivationmechanism involves the GSH-conjugation path-way. Expression of every P450 isoform studied infreshly isolated PT and DT cells decreased after48 h of primary culture. The only P450 isoformwhose expression was still detectable in primaryculture was CYP4A. The ability to detect CYP4Aand to induce its expression with ciprofibrate,suggests that these primary cultures may be goodmodel systems to study the function and regula-tion of CYP4A expression. TRI and DCVC werecytotoxic to both PT and DT cells after relativelylong periods of treatment, with DCVC beinghighly cytotoxic and TRI being only moderatelycytotoxic. Both TRI and DCVC appeared tocause a small degree of sublethal injury and regen-eration or proliferation in these cells, as evidencedby changes in DNA levels and in vimentin expres-sion, but the mechanism behind this response iscurrently unknown but is under investigation.
Acknowledgements
This work was supported by National Instituteof Diabetes and Digestive and Kidney DiseasesGrant R01-DK40725 (to L.H.L.) and through theuse of the Imaging and Cytometry Core of theEHS Center (Grant P30-ES02526), provided bythe National Institute of Environmental HealthSciences.
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