Enhanced Toxicity for Mice of Combinations of Bacterial Endotoxin with Antitumor Drugs

Home , Mercaptopurine

ANTIMrCROBIAL AGENTS AND CHEMOTHERAPY, May 1973, p. 599-606 Vol. 3, No. 5 Copyright 0 1973 American Society for Microbiology Printed in U.SA. Enhanced Toxicity for Mice of Combinations of Bacterial Endotoxin with Antitumor Drugs

NELDA M. MARECKI AND S. G. BRADLEY Department of Microbiology, Virginia Commonwealth University, Richmond, Virginia 23298 Received for publication 6 February 1973

The toxicity of Salmonella typhosa 0901W endotoxin to mice was potentiated by (per kilogram) 1 mg of colchicine, 20 mg of emetine, 100 mg of 6-mercaptopurine, 100 mg of 6-methylmercaptopurine riboside, 75 mg of methotrexate, 2 mg of sparsomycin, or 2.5 mg of vinblastine. No potentiation of endotoxin lethality was evident with simultaneously administered (per kilogram): 200 mg of cytosine arabinoside, 450 mg of dibromomannitol, 100 mg of 5-fluorouracil, 125 mg of 5-fluorouracil deoxyriboside, 8 mg of mitomycin C, 1 mg of nitrogen mustard, or 10 mg of tris(1-aziridinyl)-phosphine sulfide. With the exception of colchicine, all drugs prolonged the duration of sleep after the administration of 80 mg of hexobarbital per kg. Simultaneous injection of endotoxin with 100 mg of 6-mercaptopurine per kg, 75 mg of methotrexate per kg, or 1 mg of vincristine per kg resulted in significantly greater lethality than administration either prior to or after the drug. However when endotoxin was administered prior to 100 mg of 5-fluorouracil per kg, lethality was significantly increased. The route of administration of endotoxin and 5-fluorouracil, 6-mercaptopurine, methotrexate, or vincristine did not influence overall lethality. Pretreatment of mice with multiple doses of Escherichia coli endotoxin resulted in a significant reduction in the lethality of 6-mercaptopurine- or vincristine-endotoxin combinations, but had no influence on 5-fluorouracil- or methotrexate-endotoxin combinations. Endotoxin- pretreated mice were more susceptible to vincristine alone and more resistant to high doses of 5-fluorouracil. The lethality of 6-mercaptopurine was increased by simultaneous administration of gram-negative isolates from feces of human patients with neoplastic disease.

The increased lethality associated with com- microsomal enzymatic activity was obtained by binations of bacterial endotoxins and various measuring the duration of hexobarbital-induced drugs has been recognized for some time. Berry sleep 1 day after, or with, concurrent drug (2) found that actinomycin D potentiated the administration. effects of endotoxin, and this interaction has Based on the results of these studies and been used as a sensitive assay for endotoxin (3, those reported by Rose et al. (12, 13), 5-fluorou- 7). Karp and Bradley (5) demonstrated an racil, 6-mercaptopurine, methotrexate, and vin- interaction with both pactamycin and spar- cristine were chosen for further studies about somycin, and endotoxin. Subsequently, a num- the mechanism of action of the drug-endotoxin ber of antineoplastic agents and bacterial en- synergy. The effects of the sequence and route of dotoxin have been demonstrated to interact drug and endotoxin administration were eval- synergistically in vivo (11-13). uated. The capability of endotoxin pretreat- In this study, the ability of colchicine, cyto- ment to mitigate the synergistic interaction was sine arabinoside, dibromomannitol, emetine, examined. Gram-negative isolates from feces of 5-fluorouracil, 5-fluorouracil deoxyriboside, 6- human patients with neoplastic disease were mercaptopurine, 6-methylmercaptopurine ribo- used as a source of endotoxin for interaction side, methotrexate, mitomycin C, nitrogen with 6-mercaptopurine. mustard, sparsomycin, tris(1-azirdinyl)-phosphine sulfide, and vinblastine to enhance en- MATERIALS AND METHODS dotoxin lethality in mice was evaluated. An BALB/c male mice weighing 22 to 27 g were used in indication of the effect of each agent on hepatic all experiments. Mice were obtained from Battelle 599 600 MARECKI AND BRADLEY ANTIMICROB. AG. CHEMOTHER. Memorial Institute, Laboratory Supply Co., Inc., and (0.5 and 0.3%, respectively) broth at 37 C, harvested ARS/Sprague-Dawley. With the exception of di- by centrifugation, suspended in sterile 0.15 M NaCl, bromomannitol (NCS-94100), all drug solutions were and adjusted to an optical density of 0.3 (420 nm). prepared so that 0.01 ml per g of mouse weight would Bacterial numbers were quantitated by the pour-plate provide the desired dose when administered. Colchi- method. Colonies were counted after 48 h of incuba- cine (Nutritional Biochemicals Corp., Cleveland, tion at 37 C on peptone-yeast extract agar. The mean Ohio), cytosine arabinoside (NSC-63878), emetine lethal dose (LD50) for mice of each bacterial strain was (NSC-33669), 5-fluorouracil (NSC-19893), 5-fluoro- determined 3 days after injection. uracil deoxyriboside (NSC-27640), 6-mercaptopurine The LD50 of each drug was determined by the (NSC-755), 6-methylmercaptopurine riboside method of Reed and Muench (9) and, where applica- (NSC-40774), methotrexate (NSC-740), mitomycin C ble, by interpolation of probit plots (6). Synergy was (NSC-26980), nitrogen mustard (NSC-762), spar- determined by analysis of isobolograms. The tests for somycin (The Upjohn Company, Kalamazoo, Mich.), parallelism and calculation of potency ratios and tris(1-aziridinyl)-phosphine sulfide (thio-TEPA) confidence limits were done by the method of Litch- (NSC-6396), vinblastine (NSC-49842), and vincris- field and Wilcoxon (6). "One-tailed" Student's t tests tine (NSC-67574) were dissolved or suspended in were used to evaluate differences in duration of sterile distilled water and adjusted to pH 7. Due to the barbiturate-induced sleep between test and control low solubility of dibromomannitol, it was necessary to mice. The significance of differences between the use a stock solution of 15 mg/ml for injections (0.75 ml proportion of dead mice in the test and control groups per 25-g mouse yields 450 mg/kg). Salmonella typhosa was evaluated by the chi-square test. The acceptable 0901W lipopolysaccharide (Difco), and Escherichia level of significance was P < 0.05 in all statistical coli 026:B6 B lipopolysaccharide (Difco) were sus- tests. pended in 0.15 M NaCl. All drug and endotoxin injections were given simultaneously via the in- traperitoneal (i.p.) route unless otherwise indicated. RESULTS The duration of hexobarbital-induced sleep was The LD50 for S. typhosa 0901 endotoxin was determined after i.p. injection of 80 mg of hexobarbi- 13.5 mg (11.25-16.20) per kg by probit plot tal per kg (Winthrop Laboratories, New York, N.Y.). analysis and 13.8 mg/kg by Reed-Muench deter- The duration of sleep was defined as that interval A 4 between the loss and the restoration of the righting mination. dose of mg of endotoxin per kg reflex (the ability or disability to right themselves resulted in less than 5% lethality. The LD,0 for twice within a 10-s period). E. coli 026:B6 endotoxin was 8.1 mg/kg by Pretreatment of mice with E. coli endotoxin was Reed-Muench determination. carried out as follows: 1 mg of endotoxin per kg on day Of the 14 drugs evaluated in this study, 7 -6, 2 mg of endotoxin per kg on days -5 and -4, and markedly potentiated the lethal effects of en- 4 mg of endotoxin per kg on day -3. The mice were dotoxin (Table 1). In mice receiving simultane- challenged on day zero. ous injections of endotoxin and (per kilogram) 1 The cultures of E. coli 06, E. coli HB, Klebsiella mg of colchicine, 20 mg of emetine, 100 mg of pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa used in these studies were all isolated from 6-mercaptopurine, 100 mg of 6-methylmercap- rectal samples obtained from cancer patients, and topurine riboside, 75 mg of methotrexate, 2 mg maintained on nutrient agar (Difco). With the excep- of sparsomycin, or 2.5 mg of vinblastine, the tion of E. coli HB, all cultures were kindly supplied by potentiations of endotoxin lethality were statis- Viola Young of the National Cancer Institute. Cul- tically significant (P < 0.05) (Table 1). These tures were grown overnight in peptone-yeast extract potentiations occurred in a parallel manner

TABLE 1. Potentiation of the toxicity of S. typhosa endotoxin by antitumor drugs Percent dead Fold decrease Drug due to drug Endotoxin LD.. in endotoxin Slope of potentiation alonea LD5 None 13.5 (11.25-16.20) 1.0 1.87 (1.55-2.26) Colchicine, 1 mg/kg 1 0.41 (0.29-0.61) 32.9 1.86 (1.27-2.72)b Emetine, 20 mg/kg 10 0.17 (0.09-0.32) 79.4 7.63 (3.32-17.67) 6-Mercaptopurine, 100 mg/kg 0 0.58 (0.37-0.90) 23.2 2.11 (1.70-2.62)b 6-Methylmercaptopurine ribo- 0 0.26 (0.18-0.39) 51.9 1.54 (1.18-2.00)b side, 100 mg/kg Methotrexate, 75 mg/kg 10 1.33 (0.95-1.86) 10.15 1.72 (1.26-2.36)" Sparsomycin, 2 mg/kg 18 0.07 (0.03-0.16) 195.65 11.80 (2.27-61.36) Vinblastine, 2.5 mg/kg 0 0.77 (0.41-1.87) 17.5 3.88 (1.49-10.09) L L a Cumulative percent dead as of day that potentiation was calculated. 'Potentiated response was parallel (P < 0.05) to the dose-response curve of endotoxin alone. VOL. 3, 1973 TOXICITY OF ENDOTOXIN AND ANTITUMOR DRUGS 601 when colchicine, 6-mercaptopurine, 6-methyl- The lethality of various combinations of en- mercaptopurine riboside, or methotrexate was dotoxin and 6-mercaptopurine is presented dia- the potentiating agent. grammatically by means of an isobologram The diagrammatic presentation of data for (Fig. 2). It is obvious from such a representation the 6-mercaptopurine potentiation of endotoxin that the lethality of the various combinations lethality (Fig. 1) is representative of data from was greater than the additive effects of the which summary results were obtained (Tables corresponding drug and endotoxin combina- 1, 2, and 3). The simultaneous administration tions. of 100 mg of 6-mercaptopurine per kg with endo- The duration of hexobarbital-induced sleep toxin resulted in a 23.3-fold parallel potentiation was measured in mice receiving the drugs under of endotoxin lethality. This dose of 6-mercap- investigation as an indication of the effects of topurine resulted in less than 5% lethality. these agents on hepatic microsomal mixed func- Of the seven drugs which potentiated the tion oxidase activity (Table 4). The agents were lethal effects of endotoxin, only emetine failed given simultaneously with, or 1 day prior to, to be markedly potentiated by endotoxin. A hexobarbital. Colchicine did not prolong sleep dose of 2 mg of endotoxin per kg resulted in a (P > 0.05) when given simultaneously or 1 day 1.9-fold decrease in the LD50 of emetine. A dose prior to hexobarbital. The following drugs were of 2 mg of endotoxin per kg resulted in a 4.5-fold found to increase hexobarbital-induced sleep decrease in the LD,0 of colchicine, and 4 mg of significantly (P < 0.05) when administered endotoxin per kg resulted in a 4.1-fold and simultaneously with the barbiturate: di- 5.7-fold decrease in the LD,0 of 6-mercaptopu- bromomannitol, 5-fluorouracil, 5-fluorouracil rine and 6-methylmercaptopurine, respectively. deoxyriboside, 6-mercaptopurine, 6-methyl- A dose of 1 mg of endotoxin per kg resulted in a mercaptopurine riboside, methotrexate, mi- 2.7-fold and a 4.7-fold decrease in the LD,0 of tomycin C, and nitrogen mustard. All drugs methotrexate and sparsomycin, respectively. with the exception of colchicine and methotrex- One milligram of endotoxin per kg produced a ate (when given 1 day prior to hexobarbital) 2.5-fold decrease and 2 mg of endotoxin per kg a significantly (P < 0.05) increased the duration 3.6-fold decrease in the LD,0 of vinblastine of sleep. None of the drugs (Table 4) induced (Table 2). The potentiations by endotoxin oc- sleep when given alone. curred in a parallel manner with colchicine, In evaluating the effects of the sequence of 6-mercaptopurine, methotrexate, or vinblas- administration of drug and endotoxin on the tine. drug-endotoxin synergy, it was found that 6- mercaptopurine and 5-fluorouracil exhibited a definite time dependency (Table 5). In the case of 6-mercaptopurine, there was an increased 95 lethality only when endotoxin was given simul- 0 x taneously with the drug. In mice given 100 mg of 6-mercaptopurine per kg with 1 mg of endotoxin per kg, there was a significant (P < 0.05) increase in lethality with respect to any other sequences of administration. At a lower dose of I- endotoxin (0.5 mg/kg), the increase in lethality 0 0 was evident but not statistically significant (P > 0.05). x 50~_ A synergistic interaction of 5-fluorouracil and endotoxin was only observed when either 2 or 4 mg of endotoxin per kg was given prior to 100 5 mg of 5-fluorouracil per kg (Table 5). There was a significant (P < 0.05) increase in lethality 0.25 0.5 L0 2.5 5 ao 25 when endotoxin was given 1 day before 5-fluo- ENDOTOXIN (mg/kg) rouracil. Endotoxin does not appear to have any interaction with 5-fluorouracil when given si- FIG. 1. Potentiation of S. typhosa endotoxin le- multaneously, or after, 5-fluorouracil adminis- thality for mice by 6-mercaptopurine. The lethal tration. response of BALBIc mice to various doses of endotoxin with no 6-mercaptopurine (x), or 100 mg of The simultaneous administration of 1 mg of 6-mercaptopurine per kg (0) are plotted on a probit vincristine with either 1 or 2 mg of endotoxin scale. per kg resulted in a significant (P < 0.05) 602 MARECKI AND BRADLEY ANTIMICROB. AG. CHEMOTHER. TABLE 2. Potentiation of the toxicity of antitumor drugs by S. typhosa endotoxin

Endo- Fold de- Drug toxin Drug LD.. Drug LD.. with crease Slope of drug Slope of D(mi/kg)(mg/kg)i D L endotoxin in~~~~~~~LD5.drug response potentiation Colchicine 2 2.23 (1.63-3.08) 0.50 (0.34-0.73) 4.5 1.72 (1.39-2.13) 1.86 (1.38-2.51)a Emetine 2 27.29 14.64b 1.9 NDC ND 6-Mercaptopurine 4 220 (174-277) 53.8 (37.62-76.93) 4.1 1.58 (1.24-2.01) 1.79 (1.36-2.36)a 6-Methylmercapto- 4 175 (135-225) 30.5 (12.71-73.20) 5.7 1.43 (1.14-1.79) 4.18 (2.46-7.12) purine riboside Methotrexate 1 182 (155-213) 71.0 (57.75-87.23) 2.6 1.36 (1.21-1.52) 1.52 (0.98-2.36)a Sparsomycin 1 3.5 (2.56-3.88) 0.67 (0.49-0.92) 4.7 1.80 (1.42-2.29) 2.90 (1.87-4.50) Vinblastine 1 5.60 (4.12-7.45) 2.23 (1.33-4.06) 2.5 1.59 (1.27-1.99) 1.93 (1.17-3.18)a 2 1.55 (1.07-2.25) 3.6 1.88 (1.53-2.33)a

a Potentiated response was parallel (P < 0.05) to the response to drug alone. b Reed-Muench determination. c Not determined. significantly (P < 0.05) fewer deaths than in any other sequence of administration. However when mice were given 2 mg of endotoxin per kg, differences in deaths in the various sequences of administration were not significant (P > 0.05), except for that when endotoxin was given 1 day after methotrexate. 8 5-Fluorouracil, 6-mercaptopurine, methotrexate, and vincristine were administered si- 0 multaneously with endotoxin by using various 0 combinations of intravenous (i.v.) and i.p. in- SL44 jections (Table 6). It was found that in no instance was there a significant (P > 0.05)

2 difference in the degree of lethality when differ- ent combinations of route of adminstration were compared for any given drug and endotoxin. 30 60 90 120 150 180 210 When 2 mg of endotoxin per kg was adminis- 6-MERCAPTOPURINE (mg/kg) tered 1 day prior to 100 mg of 5-fluorouracil per kg, there was no significant (P > 0.05) differ- FIG. 2. Synergistic toxicity for mice of combina- ence in the degree of lethality associated with tions of 6-mercaptopurine with S. typhosa endotoxin. the various combinations of routes (Table 6). The dashed line connects LD.0 values for various There was no lethality associated with any of combinations; the solid line indicates the responses expected for additive effects. these drugs when given alone either i.v. or i.p. There was 10% lethality associated with i.v. administration of 2 mg of endotoxin per kg, and increase in deaths with respect to those deaths 5% associated with the i.p. administration of 2 occurring when endotoxin was given either be- mg of endotoxin per kg. fore or after endotoxin. However, there was Mice were pretreated with E. coli 026:B6 essentially no difference in the degree of lethal- endotoxin to increase resistance to the lethal ity caused by vincristine given either 1 or 2 days effects of endotoxin. Seventy-two hours after before or after endotoxin. This relationship was the final injection, mice were challenged with seen at both doses of endotoxin employed either 5-fluorouracil, 6-mercaptopurine, metho- (Table 5). trexate, or vincristine with or without subse- Simultaneous administration of 75 mg of quent endotoxin administration (Table 7). This methotrexate per kg and 1 mg of endotoxin per treatment schedule provided mice resistant to kg resulted in a significant (P < 0.05) increase 2.5 LD50-(20 mg of endotoxin per kg). The same in deaths with respect to other sequences of dose produced 100% lethality in nontreated administration (Table 5). When endotoxin was mice (Table 7). given 1 day after methotrexate, there were Pretreatment of mice with endotoxin did not VOL. 3, 1973 TOXICITY OF ENDOTOXIN AND ANTITUMOR DRUGS 603 TABLE 3. Antitumor drugs which did not enhance the toxicity of S. typhosa endotoxin

Dose Percent dead Fold decrease Drug (mg/kg) due to drug Endotoxin LD. | in endotoxin Slope of potentiation alone LD.. Cytosine arabinosidea 200 0 12.8 (10.94-14.98) 1.08 1.30 (1.03-1.51) 5-Fluorouracil deoxy- 125 0 11.3 (7.53-16.95) 1.22 1.93 (1.36-2.73) ribosidea Mitomycin Ca 8 5 3.9 (3.17-7.02) 3.54 3.84 (1.67-8.83) Nitrogen mustarda 1 5 12.0 (6.86-21.0) 1.15 1.56 (0.97-2.50) Dibromomannitolb, c 450 10 NDd ND ND 5-Fluorouracilb 100 1.7 ND ND ND Thio-TEPAb 10 2.5 ND ND ND a No potentiation of drug by endotoxin; i.e., no change in drug LD5, when given simultaneously with endotoxin at nonlethal levels. bThese agents did not alter the LD50 of endotoxin when administered simultaneously with endotoxin. Also, endotoxin did not affect the LD50 of these agents. c Dose limited by solubility. d Not determined. TABLE 4. Effect of antitumor drugs on under these conditions did not potentiate en- hexobarbital-induced sleep dotoxin's lethal action. However mice resistant to the lethal effects of endotoxin were also Time of admin- Mean resistant to the lethal effects of 200 mg of istration in percent Signifi- 5-fluorouracil per kg. In control mice, this dose Drug relation to increasea canceb hexobarbital resulted in 85% lethality. Ine There was significantly less (P < 0.05) lethal- Colchicine, 1 mg/kg Simultaneously 12.0 ity associated with 100 mg of 6-mercaptopurine 1 Day before 2.7 kg and 1 mg or 2 mg of endotoxin per kg in Cytosine arabinoside, Simultaneously -1.6 per 1000 mg/kg 1 Day before 66.14 mice pretreated with endotoxin. However, there Dibromomannitol, Simultaneously 22.3 was no increased susceptibility of pretreated 450 mg/kg 1 Day before 33.6 mice to high doses of 6-mercaptopurine. Emetine, 20 mg/kg Simultaneously 21.7 Mice resistant to the lethal effects of en- 1 Day before 40.9 5-Fluorouracil, 100 Simultaneously 33.4 dotoxin were not protected from the methotrex- mglkg 1 Day before 33.1 ate-endotoxin interaction, and they did not 5-Fluorouracil deoxy- Simultaneously 76.3 show any increased sensitivity to methotrexate riboside, 100 1 Day before 121.3 mg/kg alone. 6-Mercaptopurine, Simultaneously 65.6 Pretreatment of mice with endotoxin pro- 100 mg/kg 1 Day before 76.9 vided significant (P < 0.05) protection to the 6-Methylmercapto- Simultaneously 40.3 interaction of 1 mg of vincristine and 2 mg of purine riboside, 1 Day before 68.8 endotoxin per kg (Table 7). This protective 100 mg/kg Methotrexate, 75 Simultaneously 82.6 trend was apparent at 1 mg of vincristine and 1 mg/kg 1 Day before 7.8 mg of endotoxin per kg but was not statistically Mitomycin C, 8 mg/kg Simultaneously 44.8 significant (P > 0.05). It is of interest to note 1 Day before 85.6 that lethality in pretreated mice given 5 mg of Nitrogen mustard, 1 Simultaneously 36.9 mg/kg 1 Day before 144.2 vincristine per kg only was significantly (P < Sparsomycin, 2 Simultaneously 10.0 0.05) greater than in nontreated mice receiving mg/kg 1 Day before 65.7 the same dose of vincristine (Table 7). Thio-TEPA, 10 Simultaneously 22.3 Nonlethal doses of the following gram-nega- mg/kg 1 Day before 65.7 Vinblastine, 2.5 Simultaneously 5.6 tive bacteria were found to contain sufficient mg/kg 1 Day before 25.9 endotoxin to potentiate the lethal effects of 100 mgof 6-mercaptopurine per kg (Table 8): E. coli ' The median time of sleep of control mice receiving 80 mg HB, E. coli 06, K. pneumoniae 10, P. hexobarbital per kg was 32.5 min. E. coli 06 and P. Significance determined at P < 0.05 by a one-tailed t aeruginosa, and P. mirabilis. test. aeruginosa when given simultaneously with 6- mercaptopurine resulted in 20 and 30% lethal- alter their response to simultaneously adminis- ity, respectively. The other three cultures in tered 5-fluorouracil and endotoxin. As previ- combination with 6-mercaptopurine resulted in ously demonstrated (Table 3), 5-fluorouracil 90% lethality within 3 days after challenge. 604 MARECKI AND BRADLEY ANTIMICROB. AG. CHEMOTHER.

TABLE 5. Effect of sequence of administration of drug 5-fluorouracil deoxyriboside, mitomycin C, ni- and S. typhosa endotoxin trogen mustard, or thio-TEPA administered simultaneously with S. typhosa endotoxin does Time of endo- not result in the increased lethality of either Endotoxin toxin adminis- Percent Drug (mg/kg) tration relative dead agent (Table 3). From these results and those to drug admin- reported by Rose and Bradley (11) and Rose et istration al. (12, 13), the generalization can be made that 5-Fluorouracil, 2 2 Days before 50 antitumor agents whose primary action is in- 100 mg/kg 1 Day before 95 volved with protein or ribonucleic acid synthesis Simultaneously 10 interact in a synergistic manner with endotoxin. 1 Day after 0 Agents whose primary action is on deoxyribo- 2 Days after 10 nucleic acid (DNA) synthesis or function usually 4 2 Days before 30 do not interact with endotoxin when admin- 1 Day before 80 istered simultaneously. This position is in agree- Simultaneously 0 ment with results reported by Seyberth et al. 1 Day after 0 2 Days after 10 (15). However, in the case of the DNA inhibitor 5-fluorouracil a significant increase in lethality 6-Mercaptopurine, 0.5 2 Days before 0 in mice is observed when endotoxin is adminis- 100 mg/kg 1 Day before 0 Simultaneously 30 tered prior to drug. Reissmann et al. (10) have 1 Day after 0 reported an accentuated and prolonged granu- 2 Days after 0 locytopenia, as well as a lymphocytopenia, in

1 2 Days before 0 TABLE 6. Effect of route of administration of drug 1 Day before 0 and S. typhosa endotoxin Simultaneously 70 1 Day after 0 Route of ad- 2 Days after 0 Route of ministration Percent Drug administra- of 2 mg of deada Methotrexate, 1 2 Days before 40 tion of drug endotoxin 75 mg/kg 1 Day before 45 per kg Simultaneously 100 1 Day after 15 2 Days after 55 5-Fluorouracil, i.v. i.v. 10 100 mg/kg' i.v. i.p. 0 2 2 Days before 60 i.p. i.v. 20 1 Day before 65 i.p. i.p. 5 Simultaneously 90 .v.C i.v. 84 1 Day after 40 i.v.c i.p. 100 2 Days after 55 i.p.C i.v. 100 i.p.c i.p. 85 Vincristine, 1 2 Days before 20 1 mg/kg 1 Day before 10 Simultaneously 90 6-Mercaptopurine, i.v. i.v. 100 1 Day after 10 100 mg/kgd i.v. i.p. 90 2 Days after 20 i.p. i.v. 90 i.p. i.p. 100 2 2 Days before 60 1 Day before 65 Methotrexate, i.v. i.v. 70 Simultaneously 90 75 mg/kge i.v. 80 1 Day after 55 i.p. 2 Days after 55 i.p. i.v. 75 i.p. i.p. 85 DISCUSSION Vincristine, i.v. i.v. 100 1 mg/kgd i.v. i.p. 100 The lethality of S. typhosa endotoxin for mice i.p. i.v. 80 has been shown to be potentiated by the follow- i.p. i.p. 90 ing agents: colchicine, emetine, 6-mercaptopurine, 6-methylmercaptopurine riboside, metho- a Comparisons within any drug group were not significant (P> 0.05). trexate, sparsomycin, and vinblastine. Con- 'Percent dead calculated on day 14. versely, with the exception of emetine, the c 5-Fluorouracil administered 24 hr after S. typhosa lethality of these agents is potentiated by en- endotoxin. dotoxin. It has also been shown that cytosine d Percent dead calculated on day 5. arabinoside, dibromamannitol, 5-fluorouracil, e Percent dead calculated on day 10. VOL. 3, 1973 TOXICITY OF ENDOTOXIN AND ANTITUMOR DRUGS 605 TABLE 7. Effect of pretreatment with E. coli selected antitumor agents and endotoxin is not endotoxin related to reduced liver microsomal oxidase Percent Percent activity. This is consistent with the findings of Endo- of con- Of Filkins (4) who has found that the lysosomal Drug DosemgkDoe toxin trol treated fraction of liver homogenates possess the great- (gk)(mg/kg) mice mice est capacity to inactivate endotoxin. dead dead Because of their current clinical importance None 2 0 0 and demonstrated interaction with endotoxin, 10 75 0 5-fluorouracil, 6-mercaptopurine, methotrex- 20 100 5 ate, and vincristine have been selected for additional studies on the mechanism of their 5-Fluorouracila 100 0 0 interaction with endotoxin. 6-Mercaptopurine 200 85 0 and endotoxin, and methotrexate and en- 100 1 0 0 dotoxin significantly potentiate each other in a 100 2 0 0 parallel manner. The same pattern of potentia- 6-Mercaptopurine° 100 0 0 tion has been reported for vincristine and en- 250 30 5 dotoxin (12). This indicates that the underlying 100 1 35 5 mechanism of the synergy may involve a dualis- 100 2 80 10 tic enhancement of each agent's mode of toxic behavior. If this is a correct interpretation, it is Methotrexatec 75 0 10 reasonable to assume that the synergistic in- 150 40 60 teraction would be abolished in mice made 75 1 60 70 resistant to the effects of endotoxin. This has 75 2 90 90 been found to be the case with 6-mercaptopu- Vincristine° 1 0 10 rine and vincristine; that is, by abolishing the 5 20 90 animals' response to E. coli endotoxin, mice do 1 1 30 10 not respond to the drug-endotoxin combina- 1 2 100 0 tions. Conversely, endotoxin is no longer able to potentiate the lethal action of the drug, perhaps a Percent dead calculated 14 days postchallenge. due to more rapid clearance or detoxification of Percent dead calculated 5 days postchallenge. endotoxin in the resistant animal. c Percent dead calculated 10 days postchallenge. 5-Fluorouracil does not interact in a synergistic manner with concurrent endotoxin adminis- mice receiving endotoxin 16 to 24 h prior to tration, and pretreatment does not alter this 5-fluorouracil. However, when endotoxin was relationship. Pretreatment of mice with multi- administered 4 to 24 h after 5-fluorouracil, there ple injections of E. coli endotoxin prior to was an acceleration of regeneration of primitive challenge does not alter the methotrexate- stem cells. Endotoxin alone also augments pro- endotoxin interaction. This indicates that the duction of stem cells in normal animals. Their methotrexate-endotoxin interaction involves findings help explain the time dependency of mechanisms other than a dualistic enhancement the 5-fluorouracil-endotoxin interaction. The of toxic action. When the time of death of mice increased resistance of endotoxin-pretreated receiving combinations of methotrexate and animals to high doses of 5-fluorouracil may be due to a stimulation of stem cell production TABLE 8. Potentiation of 6-mercaptopurine by live such that subsequent 5-fluorouracil administra- gram-negative organisms tion does not cause a lethal depression of the bone marrow. 6-Mer- Percent Changes in duration of barbiturate-induced Organism Organism ~~purnnedcaptoddday by3 sleep have been used as an indication of altera- (mg/g) tions of hepatic microsomal mixed function oxidase activity, or of a general cytotoxic effect. Escherichia coli HB, 1.8 x 109 100 90 With the exception of colchicine, all agents cells/kg which potentiate the effects of endotoxin in- E. coli 06, 4.5 x 109 cells/kg 100 20 crease the duration of hexobarbital-induced Klebsiella pneumoniae, 1.4 x 1010 100 90 Also those agents which do not potentiate cells/kg sleep. Pseudomonas aeruginosa, 1.3 x 100 30 endotoxin lethality produce increases in hexo- 1010 cells/kg barbital-induced sleep, indicating that the in- Proteus mirabilis, 2.5 x 10' cells/kg 100 90 creased lethality resulting from combinations of 606 MARECKI AND BRADLEY ANTIMICROB. AG. CHEMOTHER. endotoxin is considered, it becomes apparent National Cancer Institute and Virginia Commonwealth that these mice die at the same time as mice University. receiving methotrexate alone; that is, 5 to 10 days postadministration. Mice dying from en- LITERATURE CITED dotoxin alone, or in combination with 6-mer- 1. Armstrong, D., L. S. Young, R. D. Meyer, and A. H. captopurine or vmcristine die within 72 h after Blevins. 1971. Infectious complications of neoplastic disease. Med. Clin. N. Amer. 55:729-745. administration. However, death due to either 2. Berry, L. J. 1964. Effect of endotoxins on the level of 6-mercaptopurine or vincristine occurs between selected enzymes and metabolites, p. 151-159. In M. days 3 and 7. Based on dose-response curves, Landy and W. Braun (ed.), Bacterial endotoxins. time of death, and effect of resistance to en- Rutgers University Press, New Brunswick, N.J. 3. Dowling, J. N., and H. A. Feldman. 1970. Quantitative dotoxin on the synergy, it is proposed that biological assay of bacterial endotoxins. Proc. Soc. deaths due to 6-mercaptopurine- and vincris- Exp. Biol. Med. 134:861-864. tine-endotoxin combinations are due to an en- 4. Filkins, J. P. 1971. Hepatic lysosomes and the inactiva- hancement of the lethal effects of endotoxin, tion ofendotoxin. J. Reticuloendothel. Soc. 9:480-490. 5. Karp, R. D., and S. G. Bradley. 1968. Synergistic toxicity while those deaths due to 5-fluorouracil- and of endotoxin with pactamycin or sparsomycin. Proc. methotrexate-endotoxin combinations are due Soc. Exp. Biol. Med. 128:1075-1080. to an enhancement of the lethal effects of the 6. Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified drug. method of evaluating dose-effect experiments. J. Phar- macol. Exp. Ther. 96:99-113. Debilitated patients and patients receiving 7. Pieroni, R. E., E. J. Broderick, A. Bundeally, and L. intense antitumor therapy have an increased Levine. 1970. A simple method for the quantitation of susceptibility to bacterial infections (1). Since submicrogram amounts of bacterial endotoxin. Proc. many of the clinically used drugs have been Soc. Exp. Biol. Med. 133:790-794. 8. Plaa, G. L., E. A. Evans, and C. H. Hine. 1958. Relative demonstrated to interact with bacterial en- hepatotoxicity of seven halogenated hydrocarbons. J. dotoxins, an interaction of these agents with Pharmacol. Exp. Ther. 123:224-229. gram-negative organisms has implications of 9. Reed, L. J., and H. Muench. 1938. A simple method for clinical importance. estimating fifty per cent end points. Amer. J. Hyg. 27:493-497. It has been clearly demonstrated in these 10. Reissmann, K. R., K. B. Udupa, and H. Okamura. 1970. studies that nonlethal doses of various gram- Effect of endotoxin on normal and 5-fluorouracil-sup- negative bacteria are capable of interacting in a pressed hematopoietic stem cells. Clin. Res. synergistic manner with 6-mercaptopurine, a 18:652-658. 11. Rose, W. C., and S. G. Bradley. 1971. Enhanced toxicity commonly used antineoplastic agent. It is im- for mice of combinations of antibiotics with Esche- portant to note that the levels of bacteria richia coli cells or Salmonella typhosa endotoxin. administered per kg mouse weight are compara- Infect. Immunity 4:550-555. ble to the levels of bacteria commonly present 12. Rose, W. C., S. G. Bradley, and I. P. Lee. 1972. Enhanced toxicity for mice of vincristine and other chemo- per gram of human fecal matter, and of which therapeutic agents with Salmonella typhosa endotoxin gram-negative organisms constitute the major and Pseudomonas aenJginsa. Antimicrob. Ag. Chem- group (14). Therefore, an abundant, readily other. 1:489-495. available source of endotoxin continuously con- 13. Rose, W. C., S. G. Bradley, and I. P. Lee. 1972. Potentiation ofthe toxicity of several antitumor agents fronts the patient, and a possible in vivo in- by Salmonella typhosa endotoxin. Toxicol. Appl. Phar- teraction of endogenous endotoxin and antineo- macol. 23:102-111. plastic agents must be considered in light of the 14. Rosebury, T. 1962. Distribution and development of the many adverse effects which have been noted microbiota in man, p. 314, 321, 333. In microorganisms indigenous to man. McGraw-Hill, New York. with the use of such agents. 15. Seyberth, H. W., H. Schmidt-Gayk, and E. Hackenthal. 1972. Toxicity, clearance and distribution of endotoxin ACKNOWLEDGMENTS in mice as influenced by actinomycin D, cyclohexi- Tbis research was supported by a contract, NIH-NCI- mide, a-amanitin and lead acetate. Toxicon 69-2266, between the Division of Cancer Treatment of the 10:491-500.