Reductive destruction of dacarbazine, procarbazine hydrochloride, isoniazid, and iproniazid

Reductive destruction of dacarbazine, procarbazine hydrochloride, isoniazid, and iproniazid using nickel-aluminum alloy in basic solution is described. Solutions of dacarbazine 10 mg/mL were prepared by adding dacarbazine 100 mg, citric acid 100 mg, and mannitol 50 mg to 10 mL of water. Aqueous solutions of procarbazine hydrochloride 10 mg/mL were prepared from commercially available capsules, and aqueous solutions of isoniazid 10 mg/mL and iproniazid 5 mg/mL were prepared from powdered drug. Reductive destruction of drugs was accomplished by mixing each solution with an equal volume of 1 M potassium hydroxide solution and adding 1 g of nickel-aluminum alloy for each 20 mL of basified solution. The resulting mixtures were stirred for 20 hours (96 hours for iproniazid) and analyzed by high-performance liquid chromatography and gas chromatography for the presence of residual drug and degradation products. Dacarbazine solutions were also subjected to destruction by photolysis and by oxidation using potassium permanganate in sulfuric acid, and the results were compared with those obtained by reductive destruction. All reaction mixtures were tested for mutagenicity in Salmonella strains. All drugs subjected to reductive destruction were completely degraded to the limits of detection of the assay and produced only nonmutagenic reaction mixtures. The only acceptable results for dacarbazine were obtained by the reductive destruction method. Reduction of dacarbazine, procarbazine hydrochloride, isoniazid, and iproniazid with nickel-aluminum alloy in dilute base appears to be a good method for the destruction of these toxic compounds.     

The effect of alkylating agents and other drugs on the accumulation of melphalan by murine L1210 leukaemia cells in vitro

The effect of cytotoxic and other drugs on the accumulation of melphalan by L1210 murine leukaemia cells was studied. We have confirmed that uptake is an active process competitively inhibited by l-leucine. In 36 experiments in amino acid-free medium the mean concentration of melphalan taken up was 225 pmoles/10⁶ cells. High pressure liquid Chromatographie analysis showed that the majority of the drug is present as free native melphalan. 1, 3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was the only drug that stimulated accumulation, but without significant effect on influx or efflux rates. Busulphan, chlorambucil, cyclophosphamide, interferon, methotrexate and prednisolone had no effect on accumulation after 30 min melphalan transport. Adriamycin, CCNU, methyl CCNU, mustine and vincristine all impaired melphalan accumulation as did the non-cytotoxic drugs aminophylline, chlorpromazine and ouabain. Adriamycin, aminophylline, chloropromazine, indomethacin and ouabain all reduced melphalan influx.     

An analysis of 1-(2-chloroethyl)-1-nitrosourea activity at the cellular level

The effect of five different 1-(2-chloroethyl)-1-nitrosoureas on the growth of cultured P388 cells has been analyzed in terms of physical, chemical, and kinetic parameters that are related to the mechanism of action of this class of cancer chemotherapeutic agent. This study correlates structure with activity at the cellular level by using a dose function that is related to the amount of active species, the (2-chloroethyl)diazonium ion, that is formed during the period of exposure of cells to drug rather than to the initial drug dose. 1-(2-Chloroethyl)-1-nitrosourea analogues that rapidly enter the P388 cells are shown to have the same activity relative to the amount of active species formed. When analyzed in this way, activity is not influenced by the structure of the N-3 substituent, lipophilicity, or carbamoylating activity. The agents 1-(2-chloroethyl)-1-nitrosourea (CNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) all produce a 50% cell growth inhibition at 6 to 7 microM active species formed per cell volume. Chlorozotocin required a twofold higher effective dose to produce the same toxic effect. This decreased activity is attributed to the slow uptake of the water-soluble chlorozotocin into P388 and L1210 cells relative to the rate of chlorozotocin conversion to active species in medium. The yields to 2-chloroethanol from CNU, BCNU, and chlorozotocin were shown to be the same, indicating that these agents generate the same yield of alkylating intermediate at 37 degrees C and pH 7.4.     

Cross-linking of the DNA repair protein Omicron6-alkylguanine DNA alkyltransferase to DNA in the presence of antitumor nitrogen mustards

The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here, we show that the DNA repair protein, O (6)-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between (32)P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with the guanine half-mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT (a variant containing a single point mutation in the protein's active site) was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half-mustards. HPLC-ESI (+)-MS/MS sequencing of tryptic peptides obtained from the wild-type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys (145) and Cys (150)). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-[ S-cysteinyl]ethyl)- N-(2-[guan-7-yl]ethyl)- p-aminophenylbuyric acid and N-(2-[ S-cysteinyl]ethyl)- N-(2-[guan-7-yl]ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically. Mechlorethamine-induced AGT-DNA conjugates were isolated from protein extracts of AGT-expressing CHO cells but not control cells, demonstrating that nitrogen mustards can cross-link the AGT protein to DNA in the presence of other nuclear proteins. Because AGT is overexpressed in many tumor types, further investigations of the potential role of AGT-DNA cross-linking in the antitumor and mutagenic activity of antitumor nitrogen mustards are warranted.     

Mechanism of action of the nitrosoureas—IV: Reactions of bis-chloroethyl nitrosourea and chloroethyl cyclohexyl nitrosourea with deoxyribonucleic acid

Calf thymus DNA was reacted with ¹⁴C-labeled bis-chloroethyl nitrosourea (BCNU), and chloroethyl cyclohexyl nitrosourea (CCNU), and the nature of the derivatives investigated in an enzymatic digest. In agreement with earlier studies on polyribonucleotides, evidence was obtained for the formation of 7-hydroxyethyldeoxyguanosine, 3-hydroxyethyldeoxycytidine and 3,N⁴-ethanodeoxycytidine. In addition, significant amounts of 7-aminoethylguanine were identified in the hydrolysate of DNA treated with BCNU, but not in the hydrolysate of DNA treated with CCNU. Aminoethylguanine was also formed when DNA was reacted with chloroethylamine, suggesting that BCNU produced aminoethylguanine via chloroethylamine as an intermediate. Because both BCNU and CCNU are effective antitumor agents, the formation of aminoethylguanine is probably not an important cytotoxic reaction, but it may have significance as far as mutagenic or carcinogenic activities are concerned.     

Comparison of the properties of metabolites of CCNU.

Properties of the six isomeric N-(2-chloroethyl-N′-(hydroxycyclohexyl-N-nitrosoureas, which have been identified by other investigators as metabolites of N-(2-chloroethyl-N′-cyclohexyl-N-nitrosourea (CCNU), have been compared with those of CCNU and 2-[[[(2-chloroethyl)nitrosoamino]-carbonyl]amino]-2-deoxy-D-glucose (chlorozotocin). There are significant differences in the physicochemical, chemical, and biological properties of these metabolites, and the properties of some of them are significantly different from those of CCNU and chlorozotocin. The position of the hydroxy group and the steric configuration of the compound markedly affect the alkylating and carbamoylating activities of the compounds. The metabolites having the higher alkylating activities and the lower carbamoylating activities produce lethal toxicity to mice at lower molar doses but have somewhat better therapeutic indexes. The data are consistent with the hypothesis that the biological effects observed following the administration of CCNU are due to a large extent to the major metabolites with lesser effects contributed by the minor metabolites. Some of the metabolites have slightly better therapeutic indexes against murine leukemia L1210 than CCNU and chlorozotocin, and they are more soluble in water than CCNU but are active against both intraperitoneally implanted and intracerebrally implanted L1210 leukemia. There might be some therapeutic advantage to administering one of the minor metabolites instead of CCNU or chlorozotocin to cancer-bearing animals.     

亚硝脲类化合物的急性毒性及对耐药艾氏腹水癌小鼠的疗效研究及与构效关系初探

测定了亚硝脲类化合物ＣＮＵ、ＧＡＮＵ、ＡＣＮＵ、ＧＣＮＵ、ＣＬＺ、ＰＣＮＵ、ＢＣＮＵ、ＣＣＮＵ、ＳＴＺＮ、ＭｅＣＣＮＵ、ＦＣＮＵ的小鼠ＬＤ５０分别为５．８、１８．０、２６．０、２７．０、３１．５、３８．０、４８．５、５８．０、１５０．０、１８５．０、５００．０ｍｇ·ｋｇ－１．毒性大小和治疗存活率的高低无正相关。化学结构在１－Ｎ－有脂环或杂环基团则不易产生耐药性．且毒性低疗效高；ＣＮＵ因３－Ｎ－无－ＣＨ２ＣＨ２Ｃｌ基因则毒性增加；１－Ｎ－侧链有氟原子取代其毒性可明显降低。     

Limitations of urinary mutagen assays for monitoring occupational exposure to antineoplastic drugs

The sensitivity of the Salmonella reversion test of Ames as a screen for accidental absorption of 17 antineoplastic agents by drug handlers was evaluated. Dilutions of each drug were added to agar inoculated with each of two Salmonella typhimurium strains (TA98 and TA100); control plates contained no test drug. Colonies were counted after incubation at 36 degrees C for 48 hours. The drugs were tested in the presence of a liver preparation to provide metabolic activation of mutagenicity. Urine samples collected from patients after doses of three mutagenic drugs were extracted and tested with the Ames test. For 11 of the 17 drug solutions, no mutagenic activity was seen, but many of these 11 were toxic to the organisms. The most highly mutagenic drugs were doxorubicin and cisplatin, with mechlorethamine, carmustine, dacarbazine, and cyclophosphamide exhibiting less mutagenic activity. Urine from patients treated with doxorubicin or cyclophosphamide showed mutagenicity, but the results suggested that the quantity of these drugs that would have to be absorbed to produce a definite reaction in urine is unlikely to be achieved by drug handlers who use standard precautions. Because of its lack of sensitivity and the potential effects of environmental and dietary factors on the results, this bacterial mutagenicity test should not be used routinely for detection of accidental absorption of antineoplastic drugs.     

The lethal activity and repair inhibition capacity of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea

Summary The survival response of human colorectal carcinoma cells treated in vitro for 1 h with PCNU was characterized by a threshold exponential curve, Dq = 8 g/ml (1 h) and D ₀ = 22 g/ml (1 h). Continuous treatment induced decreasing degrees of cell kill although PCNU was biologically stable in solution for at least 24 h. Cells treated with PCNU were unable to recover from potentially lethal damage but were quite capable of repairing PCNU-induced sublethal damage. Thus, PCNU with different alkylating and carbamoylating than other nitrosourea congeners had similar cytotoxic and repair inhibition capacities. Any therapeutic gain in the clinical use of PCNU must derive only from its lipophilic properties and not from its superior activity at the cellular level.Abbreviations PCNU 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea (NSC 95466) - CCNU 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (NSC 79037) - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962) cis-acid, 4-(3-(2-chloroethyl)-3-nitrosoureido)-cis-cyclohexane-carboxylic acid (NSC 153174) - MeCCNU 1-(2-chloroethyl)-3-trans-(4-methyl-cyclohexyl)-1-nitrosourea (NSC 95441) - PE plating efficiency - Dq quasithreshold dose equal to the intercept with the abscissa (at 100% survival) of the exponential part of survival curve - D ₀ mean lethal dose equal to the concentration required to reduce survival by 63% on exponential part of survival curve     

Inhibition of intracellular esterases by antitumour chloroethylnitrosoureas. Measurement by flow cytometry and correlation with molecular carbamoylation activity

Antitumour chloroethylnitrosoureas (Cnus) decompose in physiological conditions yielding alkylating species and organic isocyanates. While antitumour activity is mainly attributed to the alkylation of DNA, carbamoylation of intracellular proteins by isocyanates may also have pharmacological and toxicological relevance. We previously reported a novel dynamic flow cytoenzymological assay for esterase inhibition in intact murine cells by BCNU and related isocyanates, and proposed that this might form the basis of an assay for intracellular carbamoylation. We have now examined a wide range of Cnus, isocyanates, and alkylating agents for their ability to inhibit cellular esterases. BCNU, CCNU, their derived isocyanates, and the 4-OH metabolites of CCNU exhibited potent inhibitory activity (I50 values 5.5 x 10(-5)-7.3 x 10(-4) M). Chlorozotocin and GANU were relatively inactive (I50 much greater than 10(-2) M). ACNU, TCNU and the 2-OH metabolites of CCNU exhibited intermediate activity (I50 values 1.1 x 10(-3)-2.3 x 10(-2) M). Compounds not able to form isocyanates were essentially inactive. Poor membrane permeability was also implicated in the weak activity of chlorozotocin and GANU. There was overall a good correlation between esterase inhibition and chemical carbamoylating activity, but some particular differences were identified. We concluded that flow cytoenzymological assay appears to have the potential to provide useful measurement of intracellular protein carbamoylation by existing Cnus and novel derivatives, and also offers the advantage of cell subpopulation identification for in vivo evaluation of these agents.     

Correlations of alkylating activity and mutagenicity in bacteria of cytostatic drugs

Alkylating activity of cytostatic drugs was studied in relation to their mutagenicity and toxicity in E. coli WP2 uvrA. Four classes of directly acting cytostatic drugs were studied: nitrogen mustards (nitrogen mustard, melphalan, chlorambucil and phosphoramide mustard, a metabolite of cyclophosphamide), ethyleneimine derivatives (Thio-TEPA, TEPA and triethylenemelamine), busulfan, and halogenated nitrosoureas. The reference compounds included methyl methanesulfonate, ethyleneimine and methylnitrosourea. Guanosine alkylation was determined by fluorometry. The rate of guanosine and nitrobenzylpyridine alkylation agreed well. Nitrogen mustard derivatives and triethylenemelamine were the most potent alkylating agents among the cytostatic drugs; nitrogen mustard was 5 to 10 times more active than methyl methanesulfonate. Ethyleneimine derivatives, busulfan and the nitrosoureas were relatively weak alkylating agents. Nitrogen mustard and triethylenemelamine were the most potent mutagens to bacteria; they were also among the most toxic drugs studied.     

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(β-d-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L121o leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonucleases micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [¹⁴C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest.64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210: bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210: bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.     

In the search for new anticancer drugs. XXIII: Exploration of a predictive design for anticancer drugs of carbohydrates containing N-nitrosochloroethylamino, N-nitrosomethyl, and N-nitrosoaminoxyl components.

The spin-labeled glucose nitrosoureas 13-16, streptozotocin (18), chlorozotocin (31), streptozotocin analogues of galactosyl 24 and mannosyl 28, and their tetra-O-acetyl derivatives 25 and 29, MCNU (Cymerin, 34), and glucamine (21) were synthesized and evaluated in vivo for their anticancer activities against the murine lymphocytic leukemia P388. Compounds 13-16, 18, 24, 28, 31, and 34 possessed activities ranging from 33 to 603% increase in life span (%ILS), whereas 21, 25, and 29 were inactive (%ILS = 9 to 10). All CD2F1 male mice treated with the most active compounds (13, 14, and 34) at 20 mg/kg were alive after 30 days, whereas all mice treated with the clinical drug streptozotocin (18) and clinically tested chlorozotocin (31) succumbed. Compounds 13-16, 18, 31, and 34 were further evaluated for their antineoplastic activity against lymphoid leukemia L1210. Compounds 13 and 34 on day 60 exhibited %ILS values of 557 and 713, respectively, as compared with %ILS values of 646 and 713 for CCNU (1) and the spin-labeled SLCNU (3), respectively. The lipophilicities of 13-16, 18, 21, 24, 25, 28, 29, 31, and 34 were determined using EPR and/or UV methods. A predictive design pattern was observed, with the most active drug (34) possessing some hydrophobic property (log P = 1.24), followed by 13 (log P = 1.87) and 14 (log P = 1.81) as the more active drugs with higher hydrophobicity than 34. The clinical drugs streptozotocin (18) and chlorozotocin (31) were distinctly hydrophilic and less active. Finally, it was concluded that various scattered results of anticancer activity in the literature can be explained by a linear correlation of activities with lipophilicities.     

Hepatocyte toxicity of mechlorethamine and other alkylating anticancer drugs. Role of lipid peroxidation.

The alkylating anticancer drugs, mechlorethamine (HN2), chlorambucil, cyclophosphamide, carmustine and lomustine readily induced cytotoxicity in isolated rat hepatocytes. Hepatocyte glutathione (GSH) was depleted rapidly following addition of the drugs. Lipid peroxidation ensued following GSH depletion and before cytotoxicity occurred. Furthermore, cytotoxicity was delayed by the antioxidants butylated hydroxyanisole (BHA) and alpha-tocopherol, the ferric iron chelator desferoxamine or the radical trap 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) even when added 10 min later. HN2 was much less toxic to hepatocytes under nitrogen and caused much less lipid peroxidation than under aerobic conditions. Cytotoxicity induced by HN2 was also prevented by choline, suggesting that a choline carrier is responsible for HN2 uptake in the hepatocytes. Various sulfur compounds acted as antidotes for HN2 cytotoxicity. Thiosulfate was still effective when added 30 min after HN2. Depletion of GSH in the hepatocytes markedly increased their susceptibility to HN2. However, BHA, desferoxamine or TEMPO protected these hepatocytes from HN2. This suggests that antioxidants could prove useful in preventing the increased risk of hepatotoxicity if GSH-depleting agents are used to overcome tumor resistance to nitrogen mustards.     

Capillary Gas Chromatography and Thermionic N-P-Specific Detection of 13-Bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-Chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in Stability and Pharmacokinetic Studies

An expedient, rapid, and sensitive capillary gas chromatographic method for the analysis of l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in plasma is described. Separation of the underivatized nitrosourea compounds was performed on a 0.33-mm-i.d., 25-m fused-silica, SE-30 capillary column, and detection was carried out using a thermionic N–P-specific detector. The compounds were extracted from plasma with benzene with a yield of >87%. The assay was linear in the ranges of 0.001 to 0.5 and 0.5 to 25 µg/ml for CCNU or 0.003 to 0.50 and 0.5 to 25 µg/ml for BCNU, with correlation coefficients from 0.9914 to 0.9999 and coefficients of variation (CV) of <3.3%. Other antineoplastic agents did not interfere in the assay. The method was employed to study the pharmacokinetics of BCNU in rabbits. The plasma concentration-time curves were fit to a two-compartment model with a mean (SE) , , and total-body clearance of 2.898 (0.913) hr^–1, 0.1228 (0.0179) hr^–1, and 7.211 (2.862) liters/hr · kg, respectively. Further, the stability of BCNU and CCNU in solution was examined at different temperatures. Both compounds were stable in benzene or acetone (4 to 37°C) but labile in plasma even if refrigerated. The apparent rate constants for degradation of BCNU and CCNU were 0.09921 and 0.02853 hr^–1 at 4°C and 5.998 and 2.553 hr^–1 at 37°C, respectively.     

Inhibition of the hepatic O6-alkylguanine-DNA alkyltransferase in vivo by pretreatment with antineoplastic agents

The mammalian DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AT) is inactivated during the repair process and its activity can only be restored by de novo synthesis. We have made use of this property to determine whether and to what extent various chemotherapeutic agents alkylate DNA in the O6-position of guanine, ie. produce lesions susceptible to AT repair. Adult female Fischer rats received a single i.p. injection of a high dose (LD50) of the respective agent and, 5 hr later, a chasing dose of N-nitroso-[14C]dimethylamine (0.2 mg/kg; 4 hr survival). The amount of 7-[14C]methylguanine formed was approximately 95 mumol/mol guanine and not significantly altered by pretreatment with any of the drugs. The ratio of O6-[14C]methylguanine/7-[14C]methylguanine was 0.019 for control animals, indicating that during the observation period of 4 hr, 83% of the O6-[14C]methylguanine produced had been removed by the hepatic AT. Little or no effect was found in rats that received spirohydantoin mustard, hexamethylmelamine, cis-platinum or mitomycin C. A significant increase in the O6-/7-[14C]methylguanine ratio was found after pretreatment with AZQ (0.026) and cyclophosphamide (0.028), agents for which lesions involving the O6-position of guanine have not yet been identified. N-(2-Hydroxyethyl)-N-nitrosourea and the cytostatic haloethylinitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), and N-chloroethyl-N-hydroxyethylnitrosourea (HECNU) inhibited the hepatic AT, producing a ratio of 0.025-0.035. Considerably higher ratios of 0.059 and 0.101 were observed after administration of the methylating agents procarbazine and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), respectively. Complete saturation of the repair system (O6-/7-[14C]methylguanine ratio, 0.11) was only achieved with N-methyl-N-nitrosourea.     

Complete remissions in chemotherapy of lung cancer models induced in mice by asbestos

Chemotherapy trials were run with mice bearing transplants of carcinomas originally induced from fetal mouse lung cells by asbestos. After treatments with a nitrosourea (PCNU), mice bearing transplants of a large-cell carcinoma (ASB XIV) had complete remissions (CR) in 13 of 20 animals on a 15 mg/kg regimen, in 6 of 20 on an 8 mg/kg regimen, and in none of 10 on a 4 mg/kg regimen. With comparable total doses, treatment was most effective when PCNU was given in a few large doses. In groups where CRs occurred, continued PCNU treatment of animals without CRs prolonged survival but yielded no additional CRs. No CRs of ASB XIV occurred in 80 mice treated with eight other anticancer agents or in 50 controls injected with 0.9% NaCl solution. In mice bearing transplants of a squamous cell carcinoma (ASB XIII), treatments with PCNU were followed by CRs in 3 of 38 animals on 15 mg/kg regimens and in 3 of 28 animals on 8 mg/kg regimens. In groups of 6 mice fed a retinoid (Ro 10-9359) and treated with PCNU, CRs of ASB XIII occurred in 3 animals in each of two trials and in none in a third trial. Ro 10-9359 inhibited growth of transplants of squamous cell carcinoma LC 12 that had been induced from fetal mouse lung cells by a polycyclic hydrocarbon. In trials of four other anticancer agents vs. ASB XIII, CRs occurred only with cyclophosphamide (CPA). There were 7 CRs among 8 mice treated with CPA 100 mg/kg x 3, no CRs in 10 after 100 mg/kg x 2, one CR in 8 after 50 mg/kg x 3, and no CRs in 6 after 50 mg/kg x 4. With the 50 mg/kg x 4 regimen of CPA and 7.5 mg/kg PCNU on the same days, there were 5 CRs in 8 mice. As a single agent, aziridinylbenzoquinone (AZQ) increased life span but gave no CRs. There were CRs of ASB XIII in all of 8 mice after toxic combined therapy with PCNU and AZQ. There were no CRs in 66 control mice bearing ASB XIII.     

Cassia Senna Inhibits Mutagenic Activities of Benzo[a]-pyrene,Aflatoxin B1, Shamma and Methyl Methanesulfonate

Abstract: Ethanol extract of Senokot tablets (Cassia senna concentrate used as vegetable laxative), was found to be non-mutagenic while it inhibited the mutagenicity of benzo[a]pyrene, shamma, aflatoxin B₁ and methyl methanesulfonate in the Ames histidine reversion assay using the Salmonella typhimurium tester strain TA98. While the Senokot extract completely inhibited the mutagenicity of promutagens (i.e. metabolic activation dependent) like benzo[a]pyrene and shamma, it reduced the mutagenic activity of the direct acting mutagen methyl methanesulfonate by only 58%. The mutagen aflatoxin B₁ showed a 25-fold increase in the number of histidine revertants per plate at low concentrations (1.0–4.0 μg/plate) in the presence of metabolic activation system while at high concentrations (10.0–30.0 μg/plate) it proved to be weakly mutagenic (with a 5-fold increase in the number of histidine revertants/plate) without metabolic activation. The Senokot extract completely inhibited the mutagenic effect of low concentrations of aflatoxin B₁ in the presence of metabolic activation but not that resulting from higher concentrations without metabolic activation. The results obtained with benzo[a]pyrene, shamma and aflatoxin B₁ indicated that the antimutagenic effects of Senokot extract could be largely due to an interaction with the metabolic process involved in the activation of procarcinogens. However, the results obtained with methyl methanesulfonate suggested that factors in Senokot may also interact with direct mutagens to produce some antimutagenic effects. An ethanol extract of crude senna leaves found to be weakly mutagenic also inhibited (though less than Senokot) the mutagenic effect of benzo[a]pyrene suggesting that the antimutagenic principle is present in the complex plant material itself.     

Mutagenesis in murine spermatogonia by MOPP therapy

Male mice were given mechlorethamine (2.0 mg/kg) followed immediately or 1, 2, 4, or 8 h later by procarbazine (100 mg/kg) and vincristine (0.67 mg/kg) in a protocol that stimulated the drugs and dosages used in a single cycle of clinical MOPP therapy. Seven weeks later and continuing for the next five weeks, the mice were mated to females and the dominant lethal mutant frequencies determined in their offspring by an in vitro assay. Significant (Student's t-test, p less than 0.05) mutant frequencies were detected when the interval between drug administration was between 2 and 8 h. In another series of experiments, mice were injected with these drugs using a 4-h interval between mechlorethamine and the vincristine and procarbazine (MOPP), two cycles of MOPP separated by one week, one cycle of MOPP and 2.0 Gy x-radiation separated by one week, or 2.0 Gy x-radiation alone. When each group was assayed for its dominant lethal mutant frequency, all were found to have similar, highly significant (0.001 less than p less than 0.005) induction. Together these data suggest that stable mutagenic lesions are induced in spermatagonial stem cells by both x-radiation and chemotherapeutic drugs and the frequency with which they are produced can be approximated by a single treatment. Clinical strategies to minimize genetic effects should concentrate on the sequence and timing of drug administration on those days when the drugs are administered in combination.     

Permeability of latex and polyvinyl chloride gloves to 20 antineoplastic drugs

Permeability of latex and polyvinyl chloride (PVC) gloves to 20 injectable antineoplastic drugs was studied. Four types of gloves were evaluated: latex surgical gloves, latex examination gloves, and PVC gloves in two thicknesses. Each glove material was exposed to each drug for 90 minutes, and permeation was tested using a mutagenicity assay. Individual fingertips of thin PVC gloves and latex surgical gloves were tested for permeability at five time points (2-30 minutes) using a doxorubicin coloration assay. All drugs permeated the thin PVC gloves. Latex surgical gloves were definitely permeable to two drugs (carmustine and thiotepa) and exhibited borderline permeability to mechlorethamine hydrochloride. The thick PVC gloves were definitely permeable to four drugs (carmustine, thiotepa, mechlorethamine hydrochloride, and daunorubicin hydrochloride) and exhibited borderline permeability to two drugs (doxorubicin and mercaptopurine). The latex examination gloves were permeable to carmustine, thiotepa, mechlorethamine hydrochloride, and cyclophosphamide. Doxorubicin permeation of individual fingertips of thin PVC gloves varied in time and amount. Doxorubicin did not permeate the latex surgical glove material, but testing with thiotepa showed that individual fingertips of this material also varied in permeability. Glove thickness was a major determinant of permeability; latex surgical gloves were the least permeable and thin PVC gloves the most permeable to the antineoplastic drugs tested. Within individual gloves and glove types, time and amount of permeation were not uniform.