S. Melchionne

Tumorigenicity of acridine orange.

ACRIDINE orange, 3,6-bisdimethylamino-acridine (A.O.), exhibits a variety of effects in biological systems. Drosophila melanogaster (Clark, 1953); it also inhibits tumor induction on mouse skin in two-stage carcinogenesis (Van Duuren et al., 1969), and causes photo-dynamic inactivation of tobacco mosaic virus (Sastry and Gordon, 1966) and other viruses. Moreover, acridine orange has been shown to inhibit protein and nucleic acid biosynthesis in cell culture systems (Zelenin and Liapunova, 1964; Scholtissek and Becht, 1966). Because of these varied properties, the mode of interaction of the dye with nucleic acids, particularly DNA, has been widely studied by a variety of physical methods (Van Duuren, 1969) and several modes of binding of dye to nucleic acid have been proposed (Drummond et al., 1965). The relationship between the mode of action of carcinogens, mutagens and tumor-initiating agents is a problem of continuing interest (Trainin et al., 1964; Van Duuren and Sivak, 1968). Since little is known about the tumor-initiating and carcinogenic activity of acridine orange, it was of interest to examine these properties in mice and rats. The present report gives the results of these experiments. MATERIALS AND METHODS Animals. Mice were ICR/Ha Swiss obtained from Millerton Research Farms (Millerton, N.Y.). Females were used in all experiments. The mice were vaccinated against ectromelia at age 6 weeks and started on test at age 8 weeks. All mice were housed on sterilized wood chips in metal cages, 10 to a cage. Rats were female eastern Sprague-Dawley obtained from Blue Spruce Farms (Altamont, N.Y.). The rats were 6 weeks old and weighed 120-125 g. when testing began. They were housed in suspended wire mesh cages, 2 to a cage. Both mice and rats were fed Purina Laboratory Chow and water ad libitum. The animal rooms were temperature controlled at 22-24° C. Biological testing methods. Animals were weighed and observed monthly for the duration of the experiment. Tumors were recorded and counted at each observation. Any animal judged clinically to be in poor condition was sacrificed before the end of the experiment. All animals were examined carefully post-mortem and tumors and other lesions were excised for histological examination. Tissue sections were fixed in 10% formalin, blocked in paraffin and stained with hematoxylin and eosin. Routine sections of liver were also taken in the mouse skin treatment groups which received acridine orange repeatedly. The duration

Rapid Induction of Thymic Lymphomas by Isopropyl Methanesulfonate: A Preliminary Report

Abstract The direct-acting SN1 alkylating agent isopropyl methanesulfonate (IMS) was carcinogenic by subcutaneous injection in female Hsd:(ICR)BR mice, causing thymic lymphoid neoplasms within 7 months in at least 20 of 32 treated mice. No such neoplasms were observed in mice treated with the direct-acting SN2 methyl homolog, methyl methanesulfonate (MMS). Both the IMS-treated mice and the MMS-treated mice initially received 20 μmole of the respective compounds by sc injection once weekly; however, because of toxic effects the dose of IMS was reduced to 10 μmole per injection on the 63rd day and further reduced to 5 μmole per injection on the 120th day, after which this dose was maintained until the 202nd day when the last surviving IMS-treated mouse became moribund and was sacrificed. In 2 of the MMS-treated mice, 93% of which were alive at 288 days, tumors were observed at the site of injection, one being a papilloma and the other a subcutaneous sarcoma. IMS has not previously been implicated as a carcinogen, to our knowledge. Its induction of thymic lymphomas may conceivably be related to its ability to alklyate exocyclic oxygen atoms in the DNA of hemopoietic cells.

Carcinogenicity of Acylating Agents: Chronic Bioassays in Mice and Structure-Activity Relationships (SARC)

A small number of acylating agents are known to be carcinogenic. The structure-activity relationships and carcinogenicity (SARC) of these compounds are reviewed in this report. In addition, the results of chronic bioassays of three previously untested compounds are described. Female ICR/Ha Swiss mice, 30–50 per group, were used in all tests. The test duration was 18–22 months. Diethylcarbamyl chloride (DECC), ethyl chloroformate (ECF), and dichloroacetyl chloride (DCAC) were tested by repeated skin application, in two-stage carcinogenesis with phorbol myristate acetate (PMA) as promoter, and by repeated subcutaneous injection. Dimethylcarbamyl chloride was used as a positive control together with control groups. All three compounds showed marginally significant incidences of papillomas and carcinomas when tested as initiators. ECF and DCAC did not show skin tumorigenicity in the repeated skin application tests. DECC showed weak carcinogenicity compared to DMCC. Subcutaneous injection resulted in the same pattern of tumorigenicity. Thus, the four acylating agents showed the following decreasing order of carcinogenicity: DMCC > DECC > ECF ≅ DCAC. All four compounds hydrolyze rapidly in water, with the following decreasing order of rate of hydrolysis: DCAC > DMCC ECF ≅ DECC. Their mechanisms of hydrolysis are: DMCC, DECC, and DCAC, SN1; ECF, SN2. There is no clear correlation between rate or mechanism of hydrolysis and carcinogenicity of these acylating agents.