Comparative toxicities of cephalosporin antibiotics in a rabbit kidney ...

Vol. 32, No. 3

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1988, p. 314-318

0066-4804/88/030314-05$02.00/0 Copyright C 1988, American Society for Microbiology

Comparative Toxicities of Cephalosporin Antibiotics in a Rabbit Kidney Cell Line (LLC-RK1) P. D. WILLIAMS,t* D. A. LASKA, L. K. TAY, AND G. H. HOTTENDORF Pharmaceutical Research and Development Division, Department of Experimental Toxicology, Bristol-Myers Company, Syracuse, New York 13221

Received 21 May 1987/Accepted 2 December 1987

The rabbit kidney cell line LLC-RK, was tested for its ability to discriminate the toxicities of six cephalosporin antibiotics according to their in vivo nephrotoxic potentials in rabbits. With the exception of cephalothin, which was markedly toxic to kidney cells in vitro, a good correlation between in vitro toxicity and in vivo nephrotoxicity was obtained, yielding the following toxicity rank order: ceftazidime < cefazolin cefoperazone < cephaloglycin cephaloridine. The addition of a kidney microsomal S9 fraction to the cell cultures desacetylated cephalothin as occurs in vivo and detoxified this antibiotic, providing it with the proper toxicity relative to the other cephalosporins. When compared with parent structures, desacetylated derivatives of other cephalosporins such as cephapirin were similarly found to be less toxic to LLC-RK, cells. The acetylated cephalosporin cephaloglycin was not detoxified by the kidney S9 fraction and was desacetylated three to four times slower than cephalothin by renal esterases. Thus, the rate and extent of desacetylation of cephalosporins may play a role in their in vivo nephrotoxic potential. Our results further suggest that LLC-RK, cells will provide a useful model for evaluating the potential nephrotoxicity of new cephalosporin antibiotics before in vivo studies. An important aspect of the preclinical evaluation of cephalosporin antibiotics involves the assessment of potential nephrotoxic reactions caused by this class of therapeutic agents. The rabbit has been a popular in vivo model for nephrotoxicity assessments because of the unique sensitivity of this species to the cephalosporin cephaloridine, compared with that of other laboratory animals (2, 14). Since nephrotoxic evaluation in rabbits requires relatively large quantities of newly synthesized cephalosporins and often involves time-consuming analyses such as histopathology, the preclinical selection of cephalosporin antibiotics could be greatly facilitated by in vitro systems serving as adjuncts to in vivo studies. The evaluation of in vitro systems utilizing tissues of rabbit kidney origin are therefore of interest. In the present investigation, we chose to evaluate a rabbit kidney cell line (LLC-RK1) which was established by Hull and co-workers (6) from the kidneys of New Zealand White rabbits. Preliminary studies with cephalothin, cephaloridine, and cefazolin have indicated that this cell line may be predictive of the in vivo toxicity of cephalosporins (5). It was the purpose of this study to compare the relative toxicities of a number of cephalosporin antibiotics in LLC-RK1 cells with their respective nephrotoxic potentials in vivo. Furthermore, the role of desacetylation in the detoxification of acetylated cephalosporins was additionally investigated on the basis of previous reports that cephalothin is more toxic to renal cells in culture than its desacetylated metabolite (5).

(Roerig, Div. Pfizer Inc., New York, N.Y.), cephaloglycin (Sigma Chemical Co., St. Louis, Mo.), and cephapirin (Bristol-Myers Co., Syracuse, N.Y.). 7-Acetamidocephalosporanic acid, 7-phenylacetamidocephalosporanic acid, and all desacetylated analogs were supplied by the Pharmaceutical Product Development Department, Bristol-Myers Co. In vivo nephrotoxicity studies. Adult male New Zealand White rabbits (Hazleton Research Animals, Denver, Pa.) weighing 2.5 to 4.0 kg were randomized according to predose weight and blood urea nitrogen (BUN) levels into seven groups of four rabbits each. BUN measurements were made with a Centrifichem 600 analyzer (Baker Instruments, Allentown, Pa.). A single intravenous dose of the antibiotics was administered via the ear vein. The dose levels and concentrations for the six antibiotics were as follows: cephaloridine, 125 mg/kg at 100 mg/ml; cephaloglycin, 125 mg/kg at 50 mg/ml; cefazolin, 500 mg/kg at 200 mg/ml; cefoperazone, 500 mg/kg at 200mg/ml; cephalothin, 1,000 mg/kg at 200 mg/ml; and ceftazidime, 1,000 mg/kg at 200 mg/ml. These doses were selected on the basis of historical data available from the literature and our laboratory. A control group of four rabbits received a single intravenous injection of normal saline (at a volume comparable to that used for cephalothin and ceftazidime). Forty-eight hours after injection, the rabbits were killed by an overdose of pentobarbital. The kidneys were removed, examined macroscopically, and placed in 10% neutral buffered Formalin for subsequent histopathologic evaluation. The kidneys were sectioned in samples 6 ,um thick and were stained with hematoxylin and eosin. The tissue slides were randomized, coded numerically, and examined without knowledge of the treatment of the animals. Microscopic lesions encountered in the renal cortex were scored for acute tubular damage (necrosis) as follows: 0, not remarkable; 1, slight necrosis; 2, moderate necrosis; 3, marked necrosis; 4, severe necrosis. BUN levels were determined before dosing and at termination (48 h after dosing).

MATERIALS AND METHODS Materials. The antibiotics used for the in vivo and in vitro experiments were obtained commercially as follows: cephalothin sodium and cefazolin sodium (Eli Lilly & Co., Indianapolis, Ind.), cephaloridine and ceftazidime sodium (Glaxo, Inc., Research Triangle Park, N.C.), cefoperazone sodium * Corresponding author. t Present address: Biochemical Toxicology Department, Lilly Research Laboratories, P.O. Box 708, Greenfield, IN 46140.

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TABLE 1. Cephalosporin nephrotoxicities in rabbits' Compound

Dose (mg/kg)

Cephaloridine Cephaloglycin

125 125 500 500 1,000 1,000

Pathologyb

BUN 48 h

Predose

Cefazolin

Cefoperazone Cephalothin Ceftazidime Saline

17.8 (15-20) 20.3 (20-21) 17.8 (15-23) 17.3 (15-19) 18.0 (16-20) 18.0 (16-20) 18.0 (16-20)

31.5 70.0 27.0 19.8 16.5 14.8 16.3

(24-39) (63-78) (15-42) (13-28) (13-22) (13-18) (13-19)

Gross

Microscopic

2.6 (2-4) 2.3 (2-3) 1.0 (0-3) 1.0 (0-2) 0.5 (0-1) 0.5 (0-1) 0.5 (0-1)

3.5 (3-4) 4.0 (4-4) 1.5 (0-3) 1.3 (1-2) 0.0 (0-0) 0.0 (0-0) 0.0 (0-0)

a Groups of four rabbits, single intravenous injection; 48-h clinico/anatomic pathology shown in the mean values from four rabbits. Numbers in parentheses show the range. b Necrosis scored as follows: 0, none; 1, minimal; 2, mild; 3, moderate; 4, marked.

Cell culture. An established rabbit kidney cell line (LLCRK1; ATCC CCL106; American Type Culture Collection, Rockville, Md.) between passage numbers 240 and 250 was used in all studies. Cultures were passed at a 1:5 dilution weekly with trypsin-EDTA (1:250, vol/vol) at concentrations of 0.5% trypsin and 0.2% EDTA (GIBCO Laboratories, Grand Island, N.Y.). Maintenance medium consisted of medium 199 in Hanks balanced salt solution with 25 mM HEPES (N-2'-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 10% fetal bovine serum (GIBCO). The cells were grown in 5% CO2 at 37°C in a water-jacketed incubator. For experimentation, LLC-RK1 cells were seeded at 2 x 104 cells per well in 96-well plates (Costar, Cambridge, Mass.) and allowed to grow to confluency (approximately 24 h later). Kidney and liver S9 preparation. The 9,000 x g supernatant (S9) was prepared aseptically from the kidneys and livers of young adult male New Zealand White rabbits (Hazelton Research Animals) killed with sodium pentobarbital. The kidneys were decapsulated, and the cortices were excised and homogenized in a Teflon-on-glass homogenizer at 6,400 rpm for 5 s in 3 volumes (wt/vol) of ice-cold 0.15 M KCI (pH 7.4). The resulting suspension was centrifuged at 9,000 x g for 10 min in a Sorvall RC5C centrifuge at 4°C. The supernatant was decanted and recentrifuged at 9,000 x g for 5 min. A similar protocol was used for the preparation of an S9 fraction from rabbit liver. The resulting S9 supernatants were stored at -70°C in 1.3-ml aliquots until use. Determination of cell viability. The drugs were prepared at 120% of the highest dose in the maintenance medium described above, allowing for subsequent dilutions and addition of rabbit kidney supernatant S9. These stock solutions were filter sterilized with Millex-GS syringe filters (0.22-,um pore size; Millipore Corp., Bedford, Mass.) before dilution. A 1-ml portion of the S9 fraction containing approximately 20 mg of protein as determined by the method of Lowry et al. (7) was diluted 1:10 with a buffer containing 80 ,uM MgCl2, 0.33 mM KCl, 10 mM NaH2PO4, 0.5 mM glucose-6-phosphate, and 0.41 mM NADP. This resulting S9 mixture was then supplemented at a 1:5 dilution in the maintenance medium containing various drug concentrations and controls. Control and drug-treated cultures were incubated in the presence or absence of the S9 mixture for 48 h. Cultures without S9 contained the S9 buffer at a 1:5 dilution in the same maintenance medium. Cells were removed from the culture plates by treatment with trypsin-EDTA, and the ability of the cells to exclude the cytoplasmic stain nigrosin was used as an index of viability. Live cells were counted on a hemacytometer to generate the percent viable control, calculated as: (number of live cells in

test/number of live cells in control) x 100. Viability was also assayed by the ability of the cells to accumulate neutral red dye by pinocytosis (8). Kidney and liver (S9) metabolism of cephalothin and cephaloglycin. The in vitro metabolism of cephalothin and cephaloglycin by both kidney and liver S9 fractions was determined by incubating these two antibiotics (0.5 mg/ml) in medium 199 supplemented with 25 mM HEPES, 10% fetal bovine serum, and the appropriate S9 fraction at 37°C for up to 90 min. The reactions were stopped by the addition of 2 volumes of cold methanol. The samples were then centrifuged at 7,080 x g for 10 min, and the resulting supernatant was analyzed for the parent compound and major metabolites by high-pressure liquid chromatography. High-pressure liquid chromatographic instrumentation. High-pressure liquid chromatographic analysis of cephalothin and cephaloglycin and their metabolites was monitored at 254 nm with a Vista 5000 liquid chromatograph equipped with a variable UV detector and a Vista 402 controllerintegrator (Varian Instruments, Sunnyvale, Calif.). The separation was achieved with a reverse-phase C-18 ,uBondapak column (25 cm by 4.5 mm; Waters Associates, Inc., Milford, Mass.) with a gradient which consisted of a 20-min linear gradient from 20 to 60% methanol-10 mM dibasic potassium phosphate buffer (pH 6.8). The gradient was eluted at a solvent flow rate of 1.0 ml/min. Transmission electron microscopy. Monolayer cultures of LLC-RK1 cells were fixed in situ in 25-mm2 culture flasks (Corning Glass Works, Coming, N.Y.) as follows. Monolayers were first allowed to stand for 5 min in a 1:1 solution of medium 199 plus serum-fixative (0.5% formaldehyde and 1% gluteraldehyde in 100 mM phosphate buffer, pH 7.4) at room temperature. This solution was removed, and the flasks with cells were completely filled with fixative and sent to the Pathology Reference Laboratory, Michigan Cancer Foundation, Detroit, for analysis by electron microscopy. Statistical analysis. The dose-response curves of cephalosporin toxicity to RK cells in the presence and absence of the S9 fraction were subjected to linear regression analyses to facilitate comparisons between compounds. The concentrations producing 50% lethality of the cell cultures for the various cephalosporins were generated from the linear regression program. RESULTS The in vivo nephrotoxicities of six cephalosporin antibiotics in rabbits are displayed in Table 1. The relative nephrotoxic potentials as judged by both BUN levels and renal histopathology after 48 h of treatment are cephaloridine,

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WILLIAMS ET AL. 1.40

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FIG. 1. Effect of 0.5 mg of cephaloridine per ml on cells 48 h posttreatment (x 100).

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cephaloglycin > cefazolin, cefoperazone > cephalothin, ceftazidime. The kidney lesions in this acute model were characterized by tubular necrosis which was confined to the proximal tubule of the cortex. LLC-RK1 cells appeared epithelial in culture and displayed morphologic evidence of cellular injury when exposed to nephrotoxic cephalosporins such as cephaloridine (Fig. 1). Upon ultrastructural analysis, LLC-RK1 cells were found to exhibit polarity as well as the appearance of brush border microvilli (Fig. 2A). In addition, the presence of tight junctions (zona occludens) and desmosomes in LLC-RK, cells is characteristic of transporting epithelium (Fig. 2B). The relative toxicity of cephalosporins to LLC-RK1 cells

LLC-RK1 cells in culture in the presence or absence of a rabbit renal S9 fraction. The dose producing lethality of 50% of the cells (TD50) was determined by dose-response analysis after 48 h of exposure to the antibiotics, and the viability of the cells was assessed by the nigrosin dye exclusion method. *, Medium 199-10%o fetal bovine serum-209o rabbit kidney S9; O, medium 199-10% fetal bovine serum without S9.

is shown in Fig. 3. In the absence of a renal S9 fraction, cephalothin, cephaloridine, and cephaloglycin were the most toxic. Cefazolin and cefoperazone were comparatively less toxic, while ceftazidime was the least toxic of the cephalosporins. The supplementation of the cultures with rabbit kidney S9 fraction markedly decreased the toxicity of cephalothin such that the toxicity ranking of six cephalosporins was cephaloridine, cephaloglycin > cefazolin, cefoperazone > cephalothin, ceftazidime.

FIG. 2. (A) Transmission electron micrograph of LLC-RK1 cells showing evidence of brush border microvilli (x8,000). (B) Transmission electron micrograph of LLC-RK1 cells displaying zona occludens (Z) and large desmosome at the bottom of the figure (x 12,320).

COMPARATIVE TOXICITIES OF CEPHALOSPORINS

VOL. 32, 1988 100

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The nigrosin dye exclusion assay was compared with the neutral red uptake assay for evaluating cephalosporin toxicity to LLC-RK1 cells (data not shown). The two assays showed a good correlation, and the comparative ranking of the cephalosporins tested by both assays was cephaloridine > cefazolin > cephalothin (plus S9). The role of the acetyl moiety in the toxicity of acetylated cephalosporins was examined by comparing the toxicities of cephalothin, cephapirin, and 7-phenylacetamidocephalosporanic acid with those of their desacetylated derivatives. 7-Acetamidocephalosporanic acid, a compound similar to cephalothin but without the thiophene ring, was also included as a test compound to determine the importance of the thiophene moiety in the cytotoxicity of cephalothin. Cephalothin, cephapirin, 7-phenylacetamidocephalosporanic acid, and 7-acetamidocephalosporanic acid all exhibited marked cellular toxicity toward the LLC-RK1 cells (percent viability was 80% of control values for desacetylcephalothin and desacetylcephapirin and approximately 65% for the desacetyl derivative of 7-phenylacetamidocephalosporanic acid (Fig.

4). Since desacetylated derivatives were considerably less toxic than their parent compounds, the role of the renal S9 fraction in the desacetylation process of acetylated cephalosporins was further investigated by high-pressure liquid chromatography. The choice of cephalothin and cephaloglycin as model compounds for this experiment was made because of the totally different responses shown by these two cephalosporins toward the LLC-RK1 cells in the presence and absence of exogenously added S9 preparations (Fig. 3). Incubation of cephalothin and cephaloglycin with the renal S9 preparation yielded desacetylcephalothin (retention time, 6.0 min) and desacetylcephaloglycin (retention time, 5.4 min) as the major metabolites, respectively. Also, the increase in formation of the desacetylated derivatives was linear with respect to both time of incubation and concentration of protein added for both compounds. The ability of the renal S9 fraction to desacetylate cephalothin and cephaloglycin was also compared with that of a similar preparation of liver S9 under identical conditions of

317

incubation time and protein concentrations. No difference in the ability of either S9 preparation to desacetylate cephalothin or cephaloglycin was observed. However, perhaps the most important difference observed between the metabolism of these two cephalosporins was that the rate of desacetylation of cephaloglycin by both S9 preparations was three to four times slower than the rate for cephalothin. Thus, after a 90-min incubation, less than 25% conversion of cephaloglycin to desacetylcephaloglycin was obtained. In comparison, under identical conditions of incubation, over 70% of cephalothin was already metabolized to its desacetylated derivative. Similar differences in the rate of desacetylation of cephalothin and cephaloglycin were observed utilizing purified citrus peel acetylesterase. DISCUSSION Cephalosporin antibiotics have been evaluated for their nephrotoxic potentials in vivo in the rabbit (9, 12, 13). Utilizing the rabbit as a model, we determined the comparative nephrotoxicities of six cephalosporins in vivo (cephalothin, ceftazidime, cefoperazone, cefazolin, cephaloridine, and cephaloglycin). Despite the extensive use of the rabbit as a model to assess cephalosporin nephrotoxicity, comparative evaluations of cephalosporin toxicity in vitro utilizing tissue derived from rabbit kidney have not been extensively reported. Such systems, if predictive of the relative nephrotoxic potentials in vivo, would provide useful adjuncts to in vivo selection of new cephalosporins. Sina et al. (10) have tested several cephalosporins for toxicity to rabbit renal proximal tubules and have proposed the utility of this model in assessing nephrotoxicity. In the present study, the LLC-RK1 rabbit kidney cell line was found to be sensitive to cephalosporin antibiotics, and as reported previously, cephalothin was comparatively more toxic than anticipated based on in vivo data (5). When cultures were supplemented with a rabbit kidney S9 fraction, cephalothin was detoxified, while the toxicities of the other cephalosporins were largely unaffected. The comparative

toxicity ranking of the various cephalosporins in LLC-RK1 cells correlated well with their in vivo nephrotoxic potentials (cephaloridine, cephaloglycin > cefoperazone, cefazolin > ceftazidime, cephalothin). However, whereas the in vitro and in vivo rankings of the cephalosporins appear to parallel each other, the correlation between events occurring in vitro in LLC-RK1 cells and in vivo in kidney cells in response to cephalosporins remains to be established. Such correlations necessitate the elucidation of mechanisms of injury and cellular accumulation of cephalosporins as well as a characterization of the identity of LLC-RK1 cells. The LLC-RK1 cells do appear to possess morphologic characteristics common to renal cells in vivo including epithelial appearance, polarity, and brush border microvilli (Fig. 2). The relationship between concentrations of drugs producing toxic effects in vitro and in vivo is of additional interest in evaluating in vitro models. Wold (15) reported that renal cortical levels of cephaloridine were approximately 1.2 mg/ml after a dose of 100 mg/kg in rabbits, at which point renal function was observed to decline. The concentration of cephaloridine producing cellular damage in vitro in LLC-RK1 cells (concentration producing 50% lethality, 0.2 mg/ml) correlates well with toxic concentrations in vivo, particularly since structural damage as measured in vitro would be expected to precede functional changes, as measured in vivo by Wold

(15).

The role of desacetylation in the detoxification of cephalothin was recently proposed by (5), who demonstrated that

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WILLIAMS ET AL.

the desacetyl metabolite of cephalothin was nontoxic compared with the parent molecule. Since the desacetylation of cephalothin in vivo is well described (1, 3), this metabolic pathway may represent a detoxification process for cephalothin and other acetylated cephalosporins. To examine further the role of the acetyl moiety in the toxicity of cephalothin, we evaluated related derivatives. The marked toxicity of 7-acetamidocephalosporanic acid, a derivative of cephalothin lacking the thiophene moiety, to LLC-RK1 cells further implicates the acetyl rather than the thiophene function in the cytotoxicity of cephalothin. Cojocel et al. (4) similarly concluded that the toxicity of cephaloridine was due to the pyridinium moiety rather than the thiophene ring. In parallel, several other acetylated cephalosporins (cephapirin, 7-phenylacetamidocephalosporanic acid, cephaloglycin) were also shown to be toxic to LLC-RK1 cells (Fig. 4). As with desacetylcephalothin, the desacetyl derivatives of cephapirin and 7-phenylacetamidocephalosporanic acid showed reduced toxicities compared with the parent compounds. These results therefore support the possibility that metabolic conversion of acetylated cephalosporins to their desacetylated derivatives represents a detoxification mechanism. Thus, conditions leading to compromised desacetylation capacity in vivo may result in an increased risk of nephrotoxicity with acetylated cephalosporins. The apparent lack of detoxification of the acetylated cephalosporin cephaloglycin in vitro by the renal S9 preparation contrasts the effect obtained with cephalothin, the other acetylated cephalosporin. Results from our in vitro studies comparing the desacetylation of cephaloglycin and cephalothin by renal and liver S9 preparations suggest that cephaloglycin is not desacetylated to the same extent as other acetylated cephalosporins. Although both liver and renal S9 preparations are capable of desacetylating the two compounds, the rate for cephaloglycin is almost three to four times slower than the rate for cephalothin. These results may indicate that cephaloglycin is not as good a substrate as cephalothin for tissue esterases. Our findings are therefore in accordance with the in vivo rat data of Sullivan et al. (11). These workers reported that 24 h after oral administration of cephaloglycin to rats, only 2% was excreted in the urine as desacetylcephaloglycin. Thus, the slow rate of conversion of cephaloglycin to its desacetylated derivative could explain both the toxicity of cephaloglycin to the LLC-RK1 cells and its nephrotoxicity in vivo. In conclusion, the rabbit kidney cell line LLC-RK1 may be used to rank several cephalosporin antibiotics according to their nephrotoxic potentials in vivo. Although the correlation between in vitro and in vivo events (cytotoxicity versus nephrotoxicity) remains to be established, in vitro models such as LLC-RK1 kidney cells in culture may provide an efficient means for compound selection before in vivo eval-

ANTIMICROB. AGENTS CHEMOTHER.

uation of cephalosporin antibiotics. Furthermore, our studies provide additional evidence that the stability of the acetyl moiety is an important determinant of the toxic potential of acetylated cephalosporins such as cephalothin and cephaloglycin. LITERATURE CITED 1. Anderson, K. E. 1978. On the pharmacokinetics of cephalosporin antibiotics. Scand. J. Infect. Dis. Suppl. 13:37-46. 2. Atkinson, R. M., P. P. Currie, B. Davis, D. A. H. Pratt, H. M. Sharpe, and E. G. Tomich. 1977. Acute toxicity of cephaloridine, an antibiotic derived from cephalosporin C. Toxicol. Appl. Pharmacol. 8:398406. 3. Bergeron, M. G., B. M. Hguyen, S. Trottier, and L. Gauvreau. 1977. Penetration of cefamandole, cephalothin, and desacetylcephalothin into fibrin clots. Antimicrob. Agents Chemother. 12:628-687. 4. Cojocel, C., J. Hannemann, and K. Baumann. 1985. Cephaloridine-induced lipid peroxidation initiated by reactive oxygen species as a possible mechanism of cephaloridine nephrotoxicity. Biochim. Biophys. Acta 834:402-410. 5. Hottendorf, G. H., D. A. Laska, P. D. Williams, and S. M. Ford. 1987. The role of desacetylation in the detoxification of cephalothin in renal cells in culture. J. Toxicol. Environ. Health 22:101-111. 6. Hull, R. N., A. C. Dwyer, W. R. Cherry, and 0. J. Tritch. 1965. Development and characteristics of the rabbit kidney cell strain, LLC-RK1. Proc. Soc. Exp. Biol. Med. 188:1054-1059. 7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 8. Parish, C. R., and A. Mullbacher. 1983. Automated colorimetric assay for T cell cytotoxicity. J. Immunol. Methods 58:225-237. 9. Silverblatt, F., W. 0. Harrison, and M. Turck. 1973. Nephrotoxicity of cephalosporin antibiotics in experimental animals. J. Infect. Dis. 128(Suppl.):5367-5372. 10. Sina, J. F., C. L. Bean, J. A. Bland, J. J. MacDonald, C. Noble, R. T. Robertson, and M. 0. Bradley. 1986. An in vitro assay for cytotoxicity to proximal tubule suspensions from rabbit kidney. In Vitro Toxicol. 1:13-22. 11. Sullivan, H. R., R. E. Billings, and R. E. McMahon. 1969. Metabolism of D-cephaloglycin-14C and L-cephaloglycin-'4C in the rat. J. Antibiot. 22:27-33. 12. Tune, B. M. 1975. Relationship between the transport and toxicity of cephalosporins in the kidney. J. Infect. Dis. 132: 189-194. 13. Welles, J. S., W. R. Gibson, P. N. Harris, R. M. Small, and R. D. Anderson. 1966. Toxicity, distribution, and excretion of cephaloridine in laboratory animals, p. 863-869. Antimicrob. Agents Chemother. 1965. 14. Williams, P. D., and G. H. Hottendorf. 1986. Critical appraisal of animal models of antibiotic toxicity. Modem Anal. Antibiot. 27:495-523. 15. Wold, J. S. 1981. Cephalosporin nephrotoxicity, p. 251-266. In J. B. Hook (ed.), Toxicology of the kidney. Raven Press, New York.