The effects of the four nitro-compounds nitroglycerin, nitroprusside-Na, NaNO2 and B 744-99 were studied simultaneously on length and on cGMP-levels in isolated circular strips of bovine coronary arteries. 1. All 4 nitro-compounds concentration dependently relaxed the strips in close association with pronounced increases in cGMP-levels which preceded the mechanical responses. 2. The relaxant effects of all 4 nitro-compounds were significantly potentiated by the predominant inhibitor of cGMP-hydrolysis M & B 22,948, which also potentiated the increase in cGMP-levels of the two nitro-compounds in which it was studied (nitroglycerin and nitroprusside-Na). 3. Non-substituted cGMP and -- much stronger -- its 8 bromo-derivative also relaxed the strips and these effects were likewise potentiated by M & B 22,948. 4. When the log increase in cGMP produced by the 4 nitro-compounds were plotted against percent relaxation (probit scale) a linear and highly significant positive correlation was obtained. 5. The results provide evidence that the increases in cGMP caused by the 4 nitro-compounds studied are responsible for the smooth muscle relaxing actions of these drugs.

The concept of multiple modes of toxic action denotes that an individual chemical can induce two or more toxic effects within the same series of concentrations, for example, reactive toxicity and narcosis. It appears that such toxicity confounds the ability to develop precise predictions of mixture toxicity and makes it more difficult to clearly link a dose-additive combined effect to agents in the mixture having a single common mechanism of toxic action. This initial study of a three-part series begins to examine this issue in greater detail by testing three α-halogenated acetonitriles: (1) in sham combinations, (2) in true combinations, and (3) with a nonreactive nonpolar narcotic. Iodo-, bromo-, and chloro-derivatives of acetonitrile were selected for testing based on their electro(nucleo)philic reactivity, via the SN2 mechanism, and their time-dependent toxicity individually. Reactivity of each agent was assessed in tests with the model nucleophile glutathione (GSH). Each acetonitrile was reactive with GSH, but the nonpolar narcotic 3-methyl-2-butanone was not. In addition, toxicity of the agents alone and in mixtures was assessed using the Microtox® acute toxicity test at three time points: 15, 30, and 45 min of exposure. Each of the three agents alone had time-dependent toxicity values of about 100%, making it likely that most of the toxicity of these agents, at these times, was due to reactivity. In contrast, the nonpolar narcotic agent lacked time-dependent toxicity. In mixture testing, sham combinations of each acetonitrile showed a combined effect consistent with predicted effects for dose-addition at each time point, as did the sham combination of the nonpolar narcotic. Mixture toxicity results for true acetonitrile combinations were also consistent with dose-addition, but the acetonitrile–nonpolar narcotic combinations were generally not consistent with either the dose-addition or independence models of combined effect. Based on current understanding of mixture toxicity, these results were expected and provide a foundation for the second and third studies in the series.

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Frequently the toxicity of an organic chemical mixture is close to dose-additive, even when the agents are thought to induce toxicity at different molecular sites of action. These findings appear to conflict with the hypothesis that a strictly dose-additive combined effect will be observed for agents sharing a single molecular site of toxic action within the organism. In this study, several SN2-reactive (alpha-halogen) or S(N)Ar-reactive (halogenated dinitrobenzene) soft electrophiles were tested with a model nonpolar narcotic (NPN) to determine the toxicity of the combinations. A sham combination of the model NPN (3-methyl-2-butanone) was also tested as a positive control. The study design incorporated time-dependent toxicity (TDT) determinations at 15, 30, and 45 minutes using a Microtox (Vibrio fischeri) protocol that included testing seven duplicated concentrations for each single agent and mixture per combination. Additionally, in chemico reactivity was determined for each compound using thiol in glutathione as a model nucleophile. The model NPN alone lacked reactivity and TDT. The SN2-reactive agents individually showed varying levels of both reactivity and TDT alone, while the SNAr-reactive chemicals alone were reactive and had toxicity that was fully time-dependent between 15 and 45 minutes of exposure. Data analyses indicated that the sham combination was dose additive, as expected, whereas three of four SN2:NPN combinations showed effects close to that predicted for dose addition but with some differences. The fourth SN2:NPN combination, which included an alpha-halogen with full TDT, showed a less-than-dose-additive combined effect as did both of the SNAr:NPN pairings. By incorporating TDT values, shapes of the dose-response curves, chemical reactivity data with thiol, reactive mechanisms for the soft electrophiles, and quantitative structure activity relationship information on whether the toxicity of the individual soft electrophiles did or did not exceeded that predicted for baseline narcosis, the results suggested that the alpha-halogens elicited two toxic effects at the concentrations tested (reactivity and narcotizing effects), whereas toxicity induced by the halogenated dinitrobenzenes was essentially limited to reactive effects. Collectively, these results provide experimental evidence consistent with previous explanations as to why binary mixtures of industrial organic chemicals often show combined effects that are close to dose additive, even when the chemicals are thought to induce toxicity at different molecular sites of action.