Hydrazine effects on vertebrate cells in vitro.

This study was designed to elucidate the cellular effects of hydrazine on four established tissue culture vertebrate cell lines (rat kangaroo kidney, Xenopus toad kidney, human diploid fibroblast, and Chinese hamster cells) and primary cultures of neonatal rat myocardial cells. Cells were exposed to hydrazine in various concentrations (0.001 to IO mM) for varying time periods. The resulting growth and morphological data revealed a possible site of hydrazine action. In all cell lines tested, population growth was depressed by low concentrations of hydrazine (0.01 to 0.1 mM). Cell growth was initially depressed, but it eventually returned to normal log phase growth even when fresh hydrazine was added to the culture medium. At higher concentrations (0.5 to 2.0 mM), hydrazine was lethal. Most cell types first showed population growth depression at 0.01 mM hydrazine. but the lethal concentration varied with the cell type. Cultures treated with hydrazine yielded a significantly higher number of giant, multinucleated cells. Autoradiography studies employing [3H]thymidine that the large, multinucleated cells resulted from cell fusion. cell cultures implicated the cell surface as a possible target site. Scanning electron microscopy confirmed concentration related surface differences between control and hydrazine-treated cells. Further membrane studies examining the effects of hydrazine on the contractile and intercellular spontaneous electrical activity of myocardial cells in cul- ture indicated that hydrazine also altered these membrane-related activities in a concentra- tion and time-dependent manner

that hydrazine also altered these membrane-related activities in a concentration and time-dependent manner Hydrazine (NH,NH,) is a hydroscopic, highly polar reducing agent (Raphaelian, 1966). This reactive compound, described as the ammono analog of hydrogen peroxide, can be converted to a variety of widely used alkyl derivatives. Both hydrazine and its derivatives are used extensively in the production of photographic developers, agricultural chemicals, and pharmaceutical products. The use of hydrazine both as an oxygen scavenger in industry and as a major component in high-energy 1 The U.S. Government's right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. rocket fuel cells constitutes its major commercial uses. As a result of these applications, hydrazine and its derivatives are becoming more prevalent in the environment, and their use has been criticized as a source of biological hazard.
Previous studies have detailed the hazardous effects of hydrazine and related derivatives. Hydrazine is known to effect pyrimidine-related mutations in DNA (Brown et al., 1966;Brown, 1967;Gupta and Grover, 1970;Kak and Kaul, 1973, and it is easily derivatized into a number of detrimental agents which act as both toxins and carcinogens. The most studied of these agents include the toxins, hydralazine, which interfers with smooth muscle con-traction (McLean et al., 1978), phenylhydrazine, a hemolytic agent including anemia and Heinz body formation (Jain et al., 1978), and monomethyl hydrazine, a metabolic inhibitor (Dost et al., 1976). A carcinogenic relative of the latter derivative, dimethylhydrazine, has produced both colon and blood vessel tumors in several laboratory animals (Toth and Wilson, 1971;Toth et al., 1976;Mak and Chong, 1978;Barkla and Tutton, 1978). More specifically, some of these effects are produced in isolated cellular components only under specific conditions of treatment (Brown, 1967;Kak and Kaul, 1975). The importance of these studies may be underestimated or misinterpreted without a basic understanding of the impact such compounds have on a wide variety of cell types when tested under controlled conditions.
The present study investigates the basic cellular responses of diverse vertebrate cell types in vitro to the compound hydrazine. The results implicate the cell membrane as one of the major targets of hydranine action. The microelectrode was carefully lowered into the selected cell using a de Fonbrune pneumatic micromanipulator, and the electrical properties of the cell were recorded and analyzed according to the procedures described earlier (Kitzes and Berns, 1979). Recordings were made both before exposure to hydrazine and during the indicated times after the medium bathing the cells was replaced by medium supplemented with 0.01, 0. I, or I.0 mM hydrazine. Figure 1 demonstrates the basic doserelated growth response of four cell types to hydrazine concentrations of 0.01 to 10 mM. Although the different cell types expressed varying degrees of sensitivity toward hydrazine, all cell types showed several common responses. Hydrazine was cytotoxic to all populations tested at dosages ranging from 0.5 to 4 mrvr, depending on the cell type. At lower concentrations, hydrazine produced a dose-dependent suppression (but not complete inhibition) of population growth with 0.01 to 1.0 mM being the threshold range of response for most cell types tested. For each cell type, there appeared an optimum dose (OD) of hydrazine which initially suppressed population growth from control levels and yet allowed the treated population to recover to log phase growth. This dosage appeared to be 1 .O mM for PTK2, 1 .O mM for A6,O. 1 mM for WI38, and 0.05 mM for CH cells. The return of OD-treated populations to log phase growth implies that either the action of hydrazine on a cell may be short-lived or that nonhydrazine sensitive cells in the population are being selected for.

RESULTS
In addition to being dose dependent (Fig.  l), the growth suppression seen in both A6 and PTK, cell types appeared to be related to the length of time of hydrazine exposure. The effects caused by various exposure point throughout the experiment. Beyond lengths of 1.0 mM hydrazine on PTK, and the designated time of treatment, the A6 cells are illustrated in Fig. 2. These cul-medium was replaced at each data point tures were replenished with medium con-with fresh medium containing no hydrazine. taining fresh hydrazine at the initial expo-The net population increase observed in culsure point (arrow) and at every subsequent tures receiving fresh hydrazine containing data point up to 24 or 96 hr. The cultures medium at every point beyond induction designated "continuous" received fresh is noteworthy. It suggests either a selechydrazine-containing medium at each data tion of genetically resistant cells or a physio-logical adaptation of cells to hydrazine. It is zine (Fig. 3). The "selected" cells renot likely that the observed effect is due to sponded to hydrazine treatment with severe hydrazine breakdown in the medium; if this population growth suppression similar to were occurring, at least an initial depression the untreated "naive" cells. The difference in growth rate should be observed after between the growth rates of "selected" and exposure to each fresh hydrazine application.
"naive" cells was insignificant when com-To test for the selection of hydrazine pared to the control growth rate. There was resistant cells, OD-treated A6 cells which no appreciable difference between growth achieved log phase growth (Day 10) were rates of untreated "naive" and untreated replated and treated anew with fresh hydra-"selected" controls (data not shown).

SIEMENS, KITZES, AND BERNS
These results indicate that no genetic selection was operating in the observed recovery from hydrazine-induced growth suppression. Further attempts to detect hydrazine-induced genetic mutation in several cell lines via ouabain resistance and growth in sloppy agar were completely unsuccessful.
Besides unsuccessful mutation assays, several other experiments were performed to detect hydrazine-induced cellular anomalies which would account for the behavior of the experimental populations. Examination of earlier Coulter counter data of control and hydrazine treated populations revealed an apparent cell size increase in the experimental cultures (Fig. 4). In a typical dose response growth curve of 72hr-treated populations, the experimental cultures had a measurably larger mean cell volume than the control cultures (plots of cell populations are taken from Day 5 samples of control and 1.0 mM-treated A6 and PTK2 cells shown in Fig. I Fig. 5). This threefold increase in multinucleation resulted in each of triplicate experiments performed.
The increase in cellular multinucleation suggests that hydrazine may act to produce either abnormal mitosis resulting in multiple nuclei or cell surface alterations promoting cell fusion. Experiments were undertaken to test for both possibilities in 1.0 mMtreated cell populations. Because PTK, cells remain perfectly flat throughout mitosis (Rattner and Berns, 1974), it was possible to carefully observe mitosis by light microscopy, Studies employing still and time lapse photography revealed no difference in mitotic abnormalities between control and treated populations. However, evidence for increased cell fusion in hydrazine-treated populations implicated the cell surface as a possible target site for hydrazine action (Table 2). In a mixed population of PTK, cells having either regular or 3H-tagged nuclei, the presence of multinucleated cells containing both types of nuclei (tagged and untagged) suggested that cell fusion was occurring. There was a fivefold increase of these types of cells in the hydrazinetreated populations as compared to the con- trol populations ( Fig. 6 and Table 2). Furthermore, the quantity of these cells comprising the entire multinucleate population of a culture was significantly higher (1.5-fold) in the hydrazine-exposed cultures than in control cultures.
To further investigate membrane effects, cell surface morphologies of hydrazine-treated and control A6 cells were studied (Fig. 7). Table 3   this inverse relationship is concentration dependent, paralleling the results of the growth response curves.
Since the cell fusion data and the SEM data implicated the cell membrane as a primary site of hydrazine action, additional studies were undertaken to examine the effect of hydrazine (0.01 to 1.0 mM) upon membrane-associated electrical and contractile activities of neonatal rat myocardial ventricular cells in culture.
A typical intracellular recording of spontaneous action potentials recorded in a rhythmically contracting heart cell is shown in Fig. 8a. The resting membrane potential is approximately -60 mV, and the action potentials occur at a rate of approximately 1 per second. In Figs. 8b and c are shown typical recordings 10 and 20 min following exposure of the culture to 0.01 mM hydrazine. Three effects are apparent: (1) a depolarization of the resting membrane potential, (2) a reduction of action potential amplitude, and (3) a disruption of discharge rhythmicity.
These electrical changes were accompanied by a disruption of the rhythmic contractile behavior of the cell. However, electrical activity returned to almost normal status 30 min after cells initially received medium containing 0.01 mM hydrazine (Fig. 8d).
Exposure to 0.1 mM hydrazine (Fig. 9b) resulted in (1) a more severe reduction in membrane potential that is still evident 15 min after exposure, (2) a complete absence of action potential discharge, and (3) arrhythmic baseline activity. During this time, the cell was not contracting. At this hydrazine concentration, the cells resumed normal electrical and contractile activity after 45 min in the hydrazine-supplemented medium (Fig. SC). Cells exposed to 1.0 mM hydrazine (Fig. 10) did not recover after 45 min to 1 hr in the experimental medium. At this time (Fig. lo), only very aberrant electrical activity accompanied by occasional small and atypical contractile activity was observed. However, these cells resumed normal electrical and contractile activity 15 min after the hydrazine-containing medium was replaced with normal medium (data not shown).

DISCUSSION
The purpose of this study was to examine the basic cellular effects of hydrazine. Previous studies have linked hydrazine and its derivatives with carcinogenic and mutagenic effects. Carcinogenic effects of the hydrazine analog 1,2-dimethylhydrazine have been reported in viva in studies of rodent intestines (Barlka andTutton, 1977, 1978;Jacobs, 1977;Mak and Chong, 1978;Richards, 1977;Sunter er al. 1978;Toth et al. 1976) and blood vessels (Toth and Wilson, 1971). Hydrazine has been shown to mutate DNA from a variety of sources (Raphalian, 1966;Dave, 1977;Brown, 1967;Brown et al., 1966;Gupta and Grover, 1970;Kak and Kaul, 1975;Kimball, 1977;Kimball andHirsch, 1975, 1976;Lemontt, 1977). These effects appear as the result of the chemical treatment of selected target tissues under specific conditions. In this study, however, a diverse variety of tissue culture cell types were employed to ascertain the basic cellular effects of hydrazine. The noted hydrazine-induced growth effects shown in this study could be classified into two basic types of responses. First of all, the cells demonstrated a concentrationdependent response to hydrazine treatment (Figs. 1,8,9,10 and Table 3). In these studies, different hydrazine concentrations, usually spanning three or four orders of magnitude, caused effects ranging from imperceptible cellular changes to lethality. Within this concentration range, the cells could survive, proliferate, and function. initial depression in growth, but, upon reexposure to fresh hydrazine, they appeared to attain a normal growth rate. Likewise in heart cell cultures, the aberrant electrical and contractile effects produced by the presence of 1.0 mM hydrazine abated when these cells were washed and bathed in normal medium. In the presence of low hydrazine concentrations, treated heart cells appeared to show some recovery from the observed effects (Figs. 8,9).
The mechanism whereby treated cells rebound in the presence of fresh hydrazine is unclear. It seems possible that this "tolerance" or "recovery" may be due to an increased capacity of exposed cells to inactivate hydrazine or its effectual metabolite. The production of "tolerant" cells in the presence of hydrazine could also suggest the selection of genetically resistant cells or the alteration of an affected organelle to a hydrazine-refractory state. Regardless of the mechanism, it appears that hydrazine evokes some form of selection or tolerance. This observation is supported by data showing that entire populations, rather than a few selected cells, seem altered by hydrazine treatment (Figs. 3, 4). Furthermore, Fig. 3 indicates that, in the tissues studied, hydrazine acted in a disruptive but nonmutational manner. It therefore seems logical that hydrazine may elicit these concentration dependent and reversible responses by actively and nonmutagenically interfering with a common cellular site in a wide spectrum of cell types. The SEM and electrophysiological studies, which were done on very different cell types, suggest that the cell surface may be a common target site of hydrazine action. Furthermore, the increase of multinucleation by cell fusion seen in hydrazine-treated cultures also indicates that the cell surface is a major site of this compound's action.
The above observation is further supported by the literature in which hydrazine derivatives have been shown to elicit a wide variety of effects in studies performed on biological membranes (Balduini ef al., 1977;Barkla and Tutton, 1977;Braun and  1977; Carom, 1977;Jain and Subrahmanyam, 1978;Jain et al., 1977Jain et al., , 1978Katsumata et al., 1977;McLean et al., 1978;Tsau ete al., 1977;Walter et al., 1978;Zimmer, 1977). The observations of these numerous studies support the view that the hydrazine-induced cellular effects seen in our data stem from membrane interactions with this compound. Furthermore, it seems logical that such a strong reducing agent as hydrazine would directly affect the cell structure it first contacts.