Degradation and inactivation of antitumor drugs.

Chemical methods for the degradation of 11 antineoplastic drugs [etoposide, teniposide, bleomycin, mitomycin C, cisplatin, cis-dichloro-trans-dihydroxy-bis(isopropylamine) platinum IV (CHIP), cyclophosphamide, ifosfamide, carmustine, lomustine, and methotrexate] were investigated. The success of the degradation procedures was assessed by HPLC and degree of biological inactivation by mutagenicity assays. The most widely applicable procedure was oxidation with potassium permanganate or 5.25% sodium hypochlorite solution (bleach). Oxidation completely degraded and inactivated etoposide, teniposide, bleomycin, mitomycin C, and methotrexate. In addition, oxidation followed by nucleophilic substitution resulted in the complete degradation and inactivation of cyclophosphamide and ifosfamide. Although carmustine and lomustine were chemically degraded by treatment with acidic potassium permanganate, the resulting reaction mixtures remained mutagenic. Therefore, this procedure cannot be recommended. The platinum-containing compounds, cisplatin and CHIP, were rendered nonmutagenic by reaction with sodium diethyldithiocarbamate. These easily performed, relatively safe procedures can be used to prevent exposure to mutagenic wastes and spills in the hospital setting.

Assessment of diphenylcyclopropenone for photochemically induced mutagenicity in the Ames assay.

The photochemical conversion of diphenylcyclopropenone to diphenylacetylene has recently been reported. Diphenylcyclopropenone is used in the treatment of alopecia areata and is nonmutagenic in a limited Ames assay. We examined diphenylcyclopropenone and diphenylacetylene, as well as synthetic precursors of diphenylcyclopropenone--dibenzylketone and alpha,alpha'-dibromodibenzylketone--for mutagenicity against TA100, TA98, TA102, UTH8413, and UTH8414. All compounds were nonmutagenic except alpha,alpha'-dibromodibenzylketone, which was a potent mutagen in TA100 with and without S-9 activation. The effect of photochemical activation of diphenylcyclopropenone in the presence of bacteria demonstrated mutagenicity in UTH8413 (two times background) at 10 micrograms/plate with S-9 microsomal activation. 8-Methoxypsoralen produces a mutagenic response in TA102 at 0.1 microgram/plate with 60 seconds of exposure to 350 nm light. In vitro photochemically activated Ames assay with S-9 microsomal fraction may enhance the trapping of short-lived photochemically produced high-energy mutagenic intermediates. This technique offers exciting opportunities to trap high-energy intermediates that may play an important role in mutagenesis. This method can be applied to a variety of topically applied dermatologic agents, potentially subjected to photochemical changes in normal use.

Induction of mutations in bacteria by a fragment of DNA from herpes simplex virus type 1.

A bacterial assay was developed for the study of mutagenesis by DNA of herpes simplex viruses. The histidine mutations from two of the Ames mutagenesis tester strains were recombined into the Salmonella histidine operon of the F'8 plasmid and each was transferred to a derivative strain of E. coli C from which the resident histidine operon had been deleted. One tester strain could be reverted by a chemical mutagen which induces frameshift mutations and the other could be reverted by a mutagen which induces base-pair substitution mutations. The BamHI G fragment of herpes simplex virus type 1 was cloned in each orientation into the BamHI site of the expression vectors pUC7, pUC8 and pUC9 and were introduced into the new strains of E. coli. The pUC9 plasmid carrying the BamHI G fragment of herpes simplex virus type 1 with the G-E' site closest to the lac promoter showed a higher rate of reversion in the frameshift strain, which varied up to 39-fold greater than the background rate. Since many mutagens are carcinogenic these data suggest the existence of a mutagenic peptide of herpes simplex virus type 1 which might be involved in cell transformation.

Genotoxic evaluation of the offgassing products of particle board.

It has been recognized that people are spending more time indoors and that pollutants are being found in elevated concentrations in this environment. Because the constituents of indoor air pollution can vary relative to a large number of factors, the nature of the indoor environment is extremely difficult to study. Of the materials used in construction of buildings which can elute complex mixtures of organic compounds, products such as particle board, plywood and insulation are known to release formaldehyde into the indoor environment. We have employed a modification of the Ames Salmonella/microsome assay with both DNA repair-proficient and -deficient strains and determined that one such material, particle board, emitted mutagenic and genotoxic substances. The materials offgassing from the particle board demonstrated a dose-related response in both mutagenicity and toxicity. It was also observed that incubation at 37 degrees C produced a decrease in both endpoints which was related to time of incubation. In addition, detectable amounts of twelve other organic compounds were identified as offgassing from the incubated particle board.

Permeability of four disposable protective-clothing materials to seven antineoplastic drugs.

The permeability of four types of protective-clothing material to seven injectable antineoplastic drugs was studied. The protective materials tested were Saranex-laminated Tyvek, polyethylene-coated Tyvek, nonporous Tyvek, and Kaycel. Circles 6 cm in diameter were cut from a single garment of each material and exposed to each drug. Permeation of cisplatin, etoposide, mitomycin, cyclophosphamide, carmustine, and thiotepa was assessed by the Salmonella mutagenicity test after four hours of exposure. Doxorubicin permeation was assessed qualitatively over an eight-hour exposure period using a coloration assay. Saranex-laminated Tyvek was not permeable under the test conditions. Polyethylene-coated Tyvek was slightly permeable to thiotepa and carmustine. Nonporous Tyvek was permeable to all seven drugs, and the Kaycel garment was permeable to all of the drugs except etoposide. In no instance did permeation exceed 3.3% of the applied drug dose. Saranex-laminated Tyvek was the most protective of the barrier garments, followed closely in effectiveness by the polyethylene-coated Tyvek. Clothing made from these two Tyvek composites would allow less air flow and, therefore, would be less comfortable to wear for extended periods. Garments made of nonporous Tyvek or Kaycel would be more comfortable, but their use should be accompanied by an awareness of their potential permeability to certain antineoplastic drugs.

Genotoxicity of organic chemicals frequently found in the air of mobile homes.

The 19 chemicals most commonly detected in a study of mobile homes in Texas were tested for mutagenicity using a battery of bacterial test strains; the literature was searched to obtain additional information concerning the mutagenicity and carcinogenicity of these chemicals. Formaldehyde was found to be present in 100% of the mobile homes and at the highest mean concentration (167 ppb). The remaining organic chemicals were all present at much lower mean concentrations (less than 10 ppb) and at varying frequencies (2-95%). Of the 19 chemicals tested for mutagenicity, only formaldehyde gave a positive response. A review of the literature revealed that 4 of the chemicals tested, formaldehyde, styrene, tetrachloroethylene and benzene, have been shown to be animal and/or human carcinogens. Thus, formaldehyde is not the only genotoxin present in the air of mobile homes but because it was present in the air of all mobile homes tested at much higher concentrations than the other organic chemicals, formaldehyde should be considered one of the major potential genotoxic hazards present in the air of mobile homes.

Genotoxic classification of anticancer drugs.

The effects of bacterial DNA excision repair on the mutagenic and lethal actions of 17 injectable anticancer drugs have been used to classify them into three levels of potential risk to medical personnel who are involved in their preparation and administration.

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.

Permeability of latex and polyvinyl chloride gloves to carmustine.

Permeability of latex and polyvinyl chloride gloves to the antineoplastic agent carmustine was studied. The latex gloves were of two types: sterile surgical gloves and disposable utility gloves. Polyvinyl chloride gloves of two thicknesses (0.20 mm and 0.35 mm) were tested. Both single and double thicknesses of each material were exposed to carmustine 3.33 mg/ml in a 10% aqueous solution of ethanol, and to the solvent alone, for 5-90 minutes. Permeation of carmustine was tested using a mutagenicity assay and a chemical assay. A time-dependent increase in carmustine permeation was observed for all types of materials (both double and single thicknesses). Mean amounts of carmustine permeating single gloves at 90 minutes ranged from 53 to 86 micrograms for the mutagenicity assay and 49 to 78 micrograms for the chemical assay. Double thicknesses of glove material (especially of the thicker polyvinyl chloride) reduced the amount of drug permeation. These latex and polyvinyl chloride glove materials offer only limited protection against contact exposure to carmustine. Latex surgical gloves may be slightly less permeable than the other types tested.

Mutagenic and cytogenetic analyses of organic extracts of simulated runoffs from model coal piles.

The use of coal as a fuel in utility and other industries in the United States is increasing. Typically, these utilities store their coal outdoors in large piles, and rainfall on the piles produces a runoff containing hazardous inorganic and organic materials. Four coals of varying sulfur contents, all used for fuel in the United States, were tested. Organic materials were extracted from simulated runoffs of model coal piles and were tested for mutagenicity with a Salmonella/microsomal assay and for clastogenicity and cytotoxicity in Chinese hamster ovary cells. The extracts of the high-sulfur coals and the lignite were more mutagenic and clastogenic than extracts from the low-sulfur coal.

The effects of excision repair and the plasmid pKM101 on the induction of his+ revertants by chemical agents in Salmonella typhimurium.

Expansion of the Ames Salmonella/microsome mutagenesis test to include plasmid pKM101-bearing, excision repair-proficient derivatives permits 1) the identification of mutagens that require both factors for activity; 2) the identification of genotoxins through the enhancement of survival by excision repair; and 3) the classification of substances according to the effects of excision repair on their mutagenesis. Class I includes substances that require excision repair to effect mutagenesis. Class II contains substances whose mutagenesis is not affected by excision repair. Class III mutagens cause premutational lesions in DNA which are readily removed by excision repair. This classification scheme is suggested as a preliminary step in making a risk estimation for a mutagen.

Exposure of pharmacy personnel to mutagenic antineoplastic drugs.

The Salmonella reversion test was used to measure the mutagenic activities of urine concentrates from individuals preparing cancer chemotherapy agents for i.v. administration. Longitudinal studies were performed in which the total urine produced in 24-hr periods was collected, starting on a Sunday at 7 p.m. after a duty-free weekend and extending over an 8-day period. There was no detectable increase in mutagenic activity in the urine concentrates of three pharmacy administrators who had no contact with these drugs. All six individuals admixing drugs in open-faced, horizontal laminar flow hoods displayed a 2-fold increase in mutagenesis by the fourth day with peak values of 2.7- to 24-fold occurring on Days 5 and 6, reduced values by Day 7, and a return to the spontaneous level by Day 8. When four of the six positive individuals in the preceding experiment admixed comparable amounts of chemotherapeutic drugs in a closed-face, vertical laminar flow hood, no increase in mutagenic activity was detected in their urine concentrates over the 8-day period.

Risk of handling injectable antineoplastic agents.

Uptake of mutagenic substances by persons handling injectable antineoplastic agents was studied, and various methods of preventing such exposure were evaluated. Six persons who prepared i.v. admixtures individually collected their urine in 24-hour batches during several eight-day study periods; each prepared from 12 to 90 admixtures per working day during each period. I.V. personnel prepared admixtures in both horizontal laminar-flow hoods and vertical-flow biological-safety cabinets, and using gloves and masks. Three pharmacy personnel who did not handle any drugs served as controls. Urine was concentrated (each 24-hour sample was concentrated to about 1 ml), and the Salmonella/mammalian microsome mutagenicity test (Ames test) was used to detect mutagenic activity in the urine concentrates. Mutagenicity was observed in the urine of all personnel during periods when they prepared antineoplastic admixtures in horizontal laminar-flow hoods, both when using no protection and while wearing gloves or masks. When admixtures were prepared in the vertical-flow biological safety cabinet by personnel wearing gloves, no urine mutagenicity was detected. The control group showed no urine mutagenicity. Protective intervention methods should be used by all persons handling antineoplastic agents. Guidelines for handling these drugs are presented.

Comparative structure-genotoxicity study of three aminoanthraquinone drugs and doxorubicin.

Two aminoanthraquinone analogs 1,4-bis(2-[(2-hydroxyethyl)amino]ethylamino)-9,10-anthracenedione (HAQ) and 1,4-dihydroxy-5,8-bis(2-[(2-hydroxyethyl)amino]ethylamino]-9,10-anthracenedione (DHAQ) have been shown to possess similar therapeutic activities against experimental tumors but different toxicities to the animals. In this study, the genotoxic effects of these two drugs and a new analog, 1,4-dihydroxy-5,8-bis(2-[(2-hydroxyethyl)amino]ethylamino]-9,10-anthracenedione diacetate, were analyzed by using mammalian cell cytogenetic assays (chromosome breakage and sister chromatid exchanges) as well as bacterial mutagenesis assays. The experimental therapeutic activities of these drugs in vivo correlated well with their in vitro genetic toxicities as revealed by cytogenetic assays; i.e., the drug with the highest therapeutic activity (DHAQ) was most active in inducing chromosome damage. DHAQ was also more genotoxic than Adriamycin. In cytogenetic assays, the activities of all drugs were reduced to different degrees in the presence of a S-9 metabolic system. Dis as revealed by cytogenetic assays; i.e., the drug with the highest therapeutic activity (DHAQ) was most active in inducing chromosome damage. DHAQ was also more genotoxic than Adriamycin. In cytogenetic assays, the activities of all drugs were reduced to different degrees in the presence of a S-9 metabolic system. Dis as revealed by cytogenetic assays; i.e., the drug with the highest therapeutic activity (DHAQ) was most active in inducing chromosome damage. DHAQ was also more genotoxic than Adriamycin. In cytogenetic assays, the activities of all drugs were reduced to different degrees in the presence of a S-9 metabolic system. Discrepancies were observed between results obtained from cytogenic assays and those from mutagenesis assays. Whereas DHAQ was most active in cytogenetic studies, Adriamycin was most mutagenic or toxic. HAQ was least active cytogenetically, and this activity was not changed appreciably in the presence of metabolic enzymes. However, it was metabolically activated to a bacterial mutagen. Our studies suggest that the cytogenetic and mutagenesis assays may be sensitive to the activities of different agents and of different moieties of the same agent. The application of short-term in vitro assays to identify the chemical structures responsible for genetic toxicity and for therapeutic activities in vivo may lead to the better understanding of drug activities and to the development of more effective drugs.

A synergistic increase in Bacillus subtilis 168 transformation produced by the presence of heterologous W23 DNA.

Transformations utilizing a 168-like recipient and mixtures of homologous and heterologous DNA lead to an unexpected increase in the number of transformants when the two DNAs are in equal concentration. The absolute requirement for native heterologous DNA to produce the effect was demonstrated. The increase may be due to a helping effect analogous to that found in Streptococcus.

Requirement of a partially diploid donor for the chemical induction of mutations in transforming DNA.

In the Bacillus subtilis transformation system, in vitro chemical mutagenesis may depend upon the use of a DNA donor carrying two copies of the region of interest. Transformations involving this region of the donor DNA which has been chemically treated may lead to the recovery of new mutations or segregant products by some as yet undetermined mechanism.

Genetic effects of hydralazine.

Hydralazine and its acetone condensation product (ACP) were found to induce base-pair substitution mutations in the Salmonella/microsomal activation test system and to display genetic toxicity in the PolA+/A- test system. Incubation with a rat-liver microsomal fraction did not affect the genetic toxicity of either compound. Other derivatives of hydralazine, including the major metabolite, 3-hydroxy-methyl-s-triazolo-[3,4a]phthalazine, did not yield any evidence of genetic toxicity nor were they metabolically convertible to a toxic product. Therefore, individuals who convert hydralazine to MTP slowly, the "slow acetylators", would be expected to be at risk.