Mutagenicity to Salmonella, Drosophila and the mouse bone marrow of the human antineoplastic agent fotemustine: prediction of carcinogenic potency.

The antineoplastic agent fotemustine is shown to be a base-pair mutagen to Salmonella. Activity is more marked in the uvrB-proficient strain G46 than in the repair-deficient strain TA1535. This is consistent with its ability to cross-link DNA. Potent activity as a somatic and germ-cell mutagen to Drosophila was also observed. A potent clastogenic response was given by fotemustine in the mouse bone marrow following either oral gavage or intraperitoneal injection of a single dose of 5 mg/kg. In each of these respects it is shown to be indistinguishable from the structurally related antineoplastic agent and human carcinogen MeCCNU. It is concluded that fotemustine should be regarded as having clear potential to induce cancer in humans. Based on these data, including the preponderance of chromosome breakages over recessive lethal mutations in Drosophila, an estimated rodent carcinogenic potency (TD50) of between 15-150 mg/kg is suggested for fotemustine.

Activity of the human carcinogen MeCCNU in the mouse bone marrow micronucleus assay.

The nitrosourea mustard MeCCNU is the most recent organic chemical to be classified as a human carcinogen by IARC. MeCCNU gave a strong positive response when tested in the mouse bone marrow micronucleus assay. Activity was evident using either ip injection or oral gavage of the test chemical. These results further support the correlation between human carcinogens and their genotoxicity.

Genotoxic effects of MeCCNU on human peripheral blood lymphocytes.

MeCCNU (semustine, 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, NSC 95441) is successfully used in the treatment of various human malignancies. The drug was tested for its in vitro genotoxic effects on human peripheral blood lymphocytes. Several drug concentrations were studied considering mean plasma level achieved in patients after the receipt of MeCCNU therapy. Induction of chromosome aberrations and sister chromatid exchange frequency had shown dose-effect relationship. Reduced mitotic activity and prolonged average generation time was observed in MeCCNU treated cultures. The results suggest that 'therapeutic' MeCCNU concentrations have genotoxic effects on human peripheral blood lymphocytes.

The effect of high doses of methyl-CCNU on male and female fertility in SJL/J mice.

Single IP injection of high dose (LD 10) of methyl-CCNU administered to sexually mature male mice resulted in severe inhibition of spermatogenesis and reduction in testicular wet weight, without significant changes in plasma testosterone levels, and with hyperplasia of the interstitum, including Leydig cells, in the testis. These effects were temporary, spermatogenesis and testiculer weight recovered 50 days after treatment. Mating of the treated males with normal females demonstrated absolute sterility at 20 days and full recovery at 50 days after treatment. Administration of a single lethal dose (38 mg/kg, LD70) of methyl-CCNU to immature (25 day-old) male mice also caused severe but temporary inhibition of spermatogenesis, and mating of mice which survived the treatment and reached sexual maturity, with normal females, resulted in 80 to 100% pregnancies at 40 and 70 days after treatment, respectively. Three repeated injections of 30 mg/kg (at 10 day intervals) of methyl-CCNU to 15 day-old male mice resulted in inhibition of spermatogenesis without alteration in plasma testosterone. Ninety days after this treatment, only 40% of matings with normal females resulted in pregnancies. Female mice treated once with 20 mg/kg of methyl-CCNU, 3 days before or 7 days after mating with normal males, showed complete failure to complete pregnancy-resorption of the embryos was demonstrated in those female mice treated 7 days after mating. After a second mating trial with these two treated groups, performed 40 to 55 and 42 days after the first trial, respectively, 80 and 86% of the females delivered offspring. However, average litter size was reduced to 4 in the second group, as compared to 8 in untreated mice. There were no apparent birth defects in the offspring of methyl-CCNU treated male or female mice.

Priming with low doses of methyl-CCNU reduce the toxicity of high doses of methyl-CCNU and melphalan, and increase the lifespan of mice implanted with Lewis lung carcinoma.

Pretreatment of mice with low doses of methyl-CCNU was shown to reduce the toxicity of lethal doses of methyl-CCNU or melphalan administered one or two days following the low dose. There was an increase in survival rate, body weight, thymus and kidney wet weight. Tissue morphology was less affected in the primed mice as compared to mice receiving the high dose or a high-low dose combination. In mice implanted s.c. with Lewis lung carcinoma, priming with 5 mg kg-1 methyl-CCNU 2 days before injection of a very high (35 mg kg-1) dose significantly increased the lifespan as compared to treatment with the high dose alone or with high-low dose combination. When the dose of methyl-CCNU was further increased to 40 mg kg-1 toxic death occurred, which was, however, significantly reduced by 'priming' with the low dose given. When low-high dose combination was used twice (the high dose was given on day 7 or 9, and 18 or 20 after tumour inoculation), priming with 5 mg kg-1 (but not with 10 mg kg-1) two days prior to the high dose was beneficial in reducing toxic death (in two experiments) and either increasing lifespan or not significantly increasing it. In no case was there protection of the tumour by the low-high dose combinations.

In vivo studies on the relationship between hepatic metabolism and the renal toxicity of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU).

Administration of a single sc dose of the nephrotoxic anticancer agent 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) to rats led to a time-dependent decrease in renal function (i.e., renal slice anion accumulation, renal concentrating ability, and urinary output) which was correlated with the accumulation of carbamylating and alkylating intermediates of 14C-labeled MeCCNU that bound irreversibly to kidney protein. MeCCNU also produced a dose-dependent decrease in glutathione (GSH) preferentially in liver, but not in kidney. Pretreatment with piperonyl butoxide (PIP) decreased the renal toxicity and covalent binding of MeCCNU, and ameliorated the MeCCNU-dependent decrease in liver and kidney GSH. Radioactivity detected in the urine from the PIP-pretreated group was markedly lower than that in the MeCCNU-only group. In contrast, PIP pretreatment increased the accumulation of parent MeCCNU into fatty tissue. Pretreatment with phenobarbital (PB) increased the renal toxicity of MeCCNU. Moreover, PB pretreatment resulted in the increased alkylation of both liver and kidney macromolecules and to an increase in the urinary clearance of ethylene-labeled MeCCNU. In all experiments, modifiers of hepatic biotransformation produced changes in the level of covalent binding by ethylene-labeled MeCCNU (alkylation) which correlated with the degree of toxicity of MeCCNU in the kidney. Additional evidence supporting a role for hepatic biotransformation in the toxicity of MeCCNU was provided by an in vivo/in vitro colony-forming assay which demonstrated the presence of a cytotoxic metabolite in the bile of a MeCCNU-administered rat. These studies suggest that hepatic metabolism contributes significantly to the alkylating activity of MeCCNU in the liver and the kidney, and indicate that a liver-derived metabolite may be responsible for the renal toxicity of MeCCNU.

Comparative nephrotoxicity of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) and chlorozotocin: functional-structural correlations in the Fischer 344 rat.

1-(2-Chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) and chlorozotocin (CZ; 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose) are structurally related anticancer agents which differ by virtue of the increased water solubility, and comparatively low carbamylating activity, of CZ relative to MeCCNU. In the present study, a single sc injection of either of these chloroethylnitrosoureas was nephrotoxic to male Fischer 344 rats. However, at equimolar doses, CZ was shown to be a much more potent nephrotoxicant. A lethal 40-mg/kg dose of CZ (127 microM) initially resulted in acute tubular necrosis of the proximal tubules of the cortex, followed later by a necrosis of papillary collecting ducts. In contrast, lethal doses of MeCCNU (100-180 mg/kg; 400-730 microM) produced only minimal proximal tubule injury. A 250-mg/kg (1 mM) dose of MeCCNU resulted in massive papillary necrosis within 7 days, with only limited necrosis to the proximal tubules. Sublethal doses of either drug, resulted in a similar, chronic, progressive nephropathy which was delayed in onset and was characterized by polyuria, enzymuria, a decrease in urine concentrating ability, and in renal slice organic ion accumulation. Alterations in less sensitive indicators of renal toxicity (i.e., proteinuria, glucosuria, and elevated blood urea nitrogen) were observed no earlier than 3 to 7 days after administration of only the highest tested doses of CZ (40 mg/kg) or MeCCNU (250 mg/kg). At sublethal doses, administration of either drug resulted in karyomegaly to the collecting ducts in the renal medulla within 2 to 4 weeks. These studies demonstrate that carbamylation-mediated reactions may not be necessary for nephrotoxicity to develop following administration of this class of antitumor agent.

Chemotherapy of large bowel carcinoma--fluorouracil (FU) + hydroxyurea (HU) vs. methyl-CCNU, oncovin, fluorouracil, and streptozotocin (MOF-Strep). An Eastern Cooperative Oncology Group study.

In this prospective randomized study of initial chemotherapy for advanced measurable metastatic large bowel carcinoma, the response rate was 6/32 (19%) for FU + HU and 5/32 (16%) for MOF-Strep; the estimated median survival is 43 weeks for both treatments. Patients who received MOF-Strep experienced substantially greater vomiting and hematologic toxicity than patients who received FU + HU (p less than 0.001).

Effects of buthionine sulfoximine on the nephrotoxicity of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU).

Administration of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1 1-nitrosourea (MeCCNU; 50-500 mg/kg) to male F344 rats caused a time- and dose-related decrease of glutathione (GSH) preferentially in the liver, but not in the kidney. A 500-mg/kg dose of MeCCNU decreased liver, lung and kidney GSH by 69, 15 and 3%, respectively, 2 hr after dosing. However, MeCCNU had no effect on the ratio of GSH/oxidized GSH or on GSH reductase activity in any tissue tested. A single i.p. dose of DL-buthionine-SR-sulfoximine, an inhibitor of GSH biosynthesis, caused tissue GSH levels to decrease at a rate which reflected the biological half-life of GSH in the respective organs. The T 1/2 for GSH in kidney, liver and lung was found to be 1.5, 5 and 9 hr, respectively. MeCCNU administered s.c. to DL-buthionine-SR-sulfoximine-pretreated rats resulted in a depletion of hepatic and renal GSH concentrations which was additive to the effects of either of these treatments alone. DL-Buthionine-SR-sulfoximine also markedly increased the nephrotoxicity of MeCCNU and resulted in a hepatotoxicity not ordinarily seen when MeCCNU was administered alone. These results suggest that a reactive electrophilic intermediate may be involved in the mechanism of MeCCNU nephrotoxicity. Moreover, that renal GSH may play a protective role against MeCCNU-induced nephrotoxicity.

Nephrotoxicity of semustine.

Semustine is an investigational cancer chemotherapeutic agent in widespread use. This agent has now been documented to produce nephrotoxicity and renal failure with long-term administration. We collected 29 cases of semustine nephrotoxicity from the literature and six unpublished cases brought to the attention of the National Cancer Institute. Using these 35 cases as a data base, we have analyzed the incidence, dose and treatment duration relationships, clinical and histologic manifestations, and clinical course of semustine nephrotoxicity. In addition, we discuss the possible mechanisms of this nephrotoxicity based upon ongoing laboratory work. We conclude that there is a high risk of severe nephrotoxicity from semustine when the cumulative dose exceeds 1200 mg/m2 and that there may be considerable delay in onset of the renal dysfunction.

Biological and biochemical effects of N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl)-N-nitrosourea on two transplantable murine colon tumors.

Other investigators have reported that transplantable murine colon Tumor 26 is more sensitive than transplantable colon Tumor 38 to treatment with N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl)-N-nitrosourea. The present report presents the results of several kinds of in vivo and in vitro experiments that were performed to compare the effects of this agent upon these two tumors or upon cultured cells derived from them. In the in vivo experiments, data were also obtained for the spleens, colons, and marrow of the host animals. In the in vivo experiments, it was observed that: (a) approximately equal quantities of 14C from the 2-chloroethyl-14C-labeled agent were fixed to the DNA of the two tumors and the three host tissues following a single i.p. injection of the radioactive agent; (b) in all of the tissues examined at 24 hr after treatment, the drug caused greater inhibition of the synthesis of DNA than of the synthesis of RNA or protein, and the extents of inhibition of DNA synthesis were greater at 24 hr after treatment than at 6 hr after treatment; (c) the inhibition of DNA synthesis was slightly greater for Tumor 26 than for Tumor 38; (d) although the extents of inhibition of synthesis of DNA by Tumor 26 and by the colonic mucosa were similar at 24 hr after treatment of the animal, colonic mucosa much more quickly recovered the ability to synthesize DNA; and (e) the agent had no significant effect upon the sizes of the pools of purine and pyrimidine ribonucleoside phosphates. Cultured cells derived from the two tumors retained their tumorigenicity upon reimplantation into mice and their differential sensitivities to the agent, although approximately equal quantities of the 14C of the radioactive agent were fixed to the nuclei of the cells. In the in vitro experiments, the main effect of the agent upon the synthesis of macromolecules was the delayed inhibition of synthesis of DNA by Tumor 26 cells. Several experimental methods yielded evidence that the agent caused strand scission of the DNA of both kinds of cells, and there was more evidence of cross-linking of the DNA of Tumor 26 cells than of Tumor 38 cells. These results are consistent with the possibility that the differences in sensitivity of the two tumors to the agent are due to differences in the extents of cross-linking of the DNA. This explanation would be in agreement with the proposal suggested by other investigators who worked with other experimental systems.

Nephrotoxicity of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) in the Fischer 344 rat.

A single s.c. injection of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU; 20-140 mg/kg) resulted in rapid decreases in renal function as well as leading to a chronic progressive nephropathy in male Fischer 344 rats. Disturbances in renal function were proportional to the dose of MeCCNU administered and included impaired tubular transport of p-aminohippuric acid, a decrease in urine concentrating ability, an increase in urine pH, polyuria, proteinuria and enzymuria. The tubular accumulation of p-aminohippuric acid by kidney slices was decreased as early as 1 hr after MeCCNU administration (100 mg/kg), was maximal within 12 hr and remained depressed for at least 28 days after a single injection of either 40 or 80 mg/kg. Changes in other measures of renal function (increased lactate dehydrogenase excretion, alkalinuria and decreased urine concentrating ability) were delayed from 1 to 6 days after MeCCNU administration and in some cases progressed in severity throughout the 28-day duration of the experiment. The delay between the first evidence of renal damage (decreased p-aminohippuric acid uptake) and the subsequent appearance of enzymuria, proteinuria, polyuria and alkalinuria appears to correspond to a similar delay between the initial insult and the eventual development of cellular necrosis and other histopathological changes. These results demonstrate that MeCCNU is a nephrotoxicant in rats and indicate that even a single acute dose may lead to chronic and irreversible effects on the kidney. The in vivo toxicity model defined herein appears to be an appropriate one for further study of the mechanism of nephrotoxicity of MeCCNU.

Effects of prochlorperazine on experimental nephrotoxicity.

In early studies of the antitumor drug 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1nitrosourea (methyl-CCNU), animal models consistently predicted that the compound would be nephrotoxic in humans. Nephrotoxicity in cancer patients who had received methyl-CCNU was not confirmed until about 6 years after clinical trials began. We have investigated the possibility that prochlorperazine, a commonly used antiemetic, might affect the development of nephrotoxicity. Prochlorperazine (1, 2, 5, and 8 mg/kg IP on days 1-3) produced a dose-related reduction in the concentrations of plasma urea nitrogen in mice that received nephrotoxic doses of methyl-CCNU (42, 52, or 63 mg/kg IP on day 1). The frequency and severity of renal lesions evaluated histopathologically were reduced significantly as the prochlorperazine dose increased. To study further this apparent protective activity of prochlorperazine, we chose a second nephrotoxin, mercuric chloride (HgCl2, 1 mg/kg IP on day 1) and a rodent species used more commonly as a model for nephrotoxicity, the rat. Prochlorperazine (2.5 or 10 mg/kg IP on days 1-5) inhibited HgCl2-induced urinary excretion of N-acetylglucosaminidase and leucine aminopeptidase. Urinary excretion of these enzymes on day 1 reflected proximal tubular epithelial degeneration and necrosis in rats that received HgCl2 alone. The severity of HgCl2-induced renal lesions evaluated histopathologically on day 16 was significantly reduced by combination treatment with prochlorperazine. Phenothiazines have numerous pharmacologic properties that might account for this observation, and additional studies will be required to establish the mechanism of this protective effect of prochlorperazine against acute nephrotoxicity in rodents.

Phase I-II study of ftorafur and methyl-CCNU in advanced colorectal cancer.

The combination of Ftorafur (NSC-148958) and methyl-CCNU (NSC-95441) was evaluated in 36 patients with advanced colorectal cancer. The principle toxicities encountered were myelosuppression, gastrointestinal, and neurological. There were no complete responses and only 5/34 (14.7%) patients achieved a partial response. Methyl-CCNU and Ftorafur does not appear to be an effective combination in advanced adenocarcinoma of the colon and rectum.