the interactive effects of alcohol and cocaine on maternal and fetal toxicity in the long-evans rat

Neurotoxicology and Teratology, Vol. 10, pp. 355-361. © Pergamon Press plc, 1988. Printed in the U.S.A. 0892-0362/88 $3.00 + .00

The Interactive Effects of Alcohol and Cocaine on Maternal and Fetal

Toxicity in the Long-Evans Rat

M I C H A E L W. C H U R C H 1

Fetal Alcohol Research Center, Department of" Obstetrics~Gynecology School o f Medicine, Wayne State University, Detroit, MI

B A R B A R A A. D I N T C H E F F

Research Institute on Alcoholism New York State Division o f Alcoholism and Alcohol Abuse, Buffalo, N Y

A N D

P E T E R K. G E S S N E R

Department o f Pharmacology and Therapeutics, School o f Medicine State University o f New York at Buffalo, Buffalo, N Y

(

R e c e i v e d 10 S e p t e m b e r 1987

CHURCH, M. W., B. A. DINTCHEFF AND P. K. GESSNER. The interactive effects of alcohol and cocaine on maternal and fetal toxicity in the Long-Evans rat. NEUROTOXICOL TERATOL 10(4) 355-361, 1988.--The number of obstetric patients with polydrug abuse problems has increased substantially in recent years. One of the most common drug combinations is alcohol and cocaine. The effect of this drug combination on pregnancy is, therefore, of interest. Consequently, the present study investigated the relative and interactive effects of these two drugs on pregnancy outcome in an animal model. Alcohol and cocaine were administered, both separately and in combination, to separate groups of pregnant Long-Evans rats from gestation day 7-19. Animals were then sacrificed and examined on gestation day 20. Control animals were given vehicle only or were nontreated. The isoboiographic method was used to evaluate the effects of the alcohol-by-cocaine interaction on select maternal and fetal variables. This method of analysis indicated that alcohol and cocaine had interactive effects that were linearly additive for some variables and infraadditive for others. In general, the results suggest that the alcohol-plus-cocaine drug combination poses a greater risk to pregnancy than either drug alone.

Alcohol Cocaine Fetal edema Fetal fatality Maternal fatality Pregnancy/pregnant Prenatal Rat Teratogenic/teratology

IN recent years, polydrug abuse has become a major public health concern. According to one recent survey, the most common drug combination among the youth is alcohol and marijuana, followed by alcohol and cocaine [21]. In terms of cocaine abusers, 85-100% report that they also drink alcohol with about 40% reporting that they drink alcohol to the point of abuse [19,39].

The combined effects of alcohol and marijuana on pregnancy in the rats has been explored [3], but the combined effects of alcohol and cocaine on pregnancy are unknown. Yet both alcohol and cocaine cross the placenta, and either drug can adversely affect pregnancy outcome. For example, alcohol can induce fetal hypoxia [29], fetal edema [30,31], and fetal hemorrhaging [27, 30, 31], as well as abruptio

1Requests for reprints should be addressed to Michael W. Church, MI 48201.

placentae, stillbirths, embryonic resorptions, premature labor, low birth weight, low maternal weight gain, and physical and behavioral anomalies in the offspring (for reviews, see [1, 2, 33].) Several recent human and animal studies suggest that maternal cocaine use during pregnancy has a number of similar effects [6, 8, 11-15, 17, 20, 23, 25, 26, 35, 40]. While many of these studies were clouded by various confounding variables (e.g., polydrug abuse, poor nutrition, toxic adulterants), a recent animal study by our laboratory has confirmed that prenatal cocaine exposure can cause abruptio placentae, stillbirths, fetal edema, fetal hemorrhaging, poor fetal and maternal weight gain, and occasional physical malformations in the offspring [15].

Since alcohol and cocaine seem to have many similar ef-

Ph.D., Fetal Alcohol Research Center, 275 East Hancock St., Detroit,

355

356 CHURCH, D I N T C H E F F AND GESSNER

fects on pregnancy outcome and since the combined use of these drugs is becoming increasingly common, the interactive effects of these drugs on pregnancy outcome are of interest. Consequently, the present study sought to evaluate the relative and interactive effects of alcohol and cocaine on standard pregnancy outcome variables, using the Long- Evans rat as an animal model. Alcohol has the well-known ability to potentiate the effects of other drugs [34]. For example, alcohol enhances cocaine-induced hepatotoxicity [36], increases the brain-to-plasma cocaine concentration ratio [38], and enhances marijuana-induced fetotoxicity [3]. We hypothesized, therefore, that alcohol and cocaine would have additive or synergistic (i.e., more than additive or ~'supraadditive") effects with regards to maternal and fetal toxicity.

METHOD

One hundred thirty timed-pregnant nulliparous female Long-Evans hooded rats, aged 90 days at the time of mating, were housed in Plexiglas cages (45x23x20 cm) with bed- dings of wood shavings. The animal rooms were temperature (22---1°C) and humidity (40--50%) controlled with a timed light cycle of 14 hr of light per day (5:00 a.m. to 7:00 p.m.).

Matching for weights, animals were assigned to one of three vehicle control groups, a nontreated control group, or one of several drug treatment groups. The morning on which a sperm plug was found was designated gestation day 0. Starting on gestation day 7 and continuing to gestation day 19 (inclusive), the rats in the drug (or vehicle) treatment groups were injected with the appropriate substance. Gestation days 7-19 include the major periods of organogenesis and central nervous system (CNS) development in the rat [37].

Dose-response studies were initially conducted for alcohol and cocaine alone [15,18]. Based on the resultant dose-response data, subsequent alcohol-plus-cocaine drug combinations were chosen. Pregnant females in the six cocaine groups were given subcutaneous injections of either 40, 50, 60, 70, 80, or 90 mg/kg/day cocaine hydrochloride (HC1) dissolved in distilled water (2% solution). Cocaine doses were selected on the basis of the literature and the desire to choose dose levels that would adequately span the range of fetal and maternal lethality [14, 15, 17, 20, 26, 28]. As a vehicle and handling control, one group of animlas was given normal saline (SC), isovolumeric to the 90 mg/kg/day group.

Pregnant females in the six alcohol groups were given injections (PO) or either 4000, 6000, 65000, 7000, 8000, or 9000 mg/kg/day of ethanol in distilled water (15% solution). Alcohol doses were selected on the basis of the literature and the desire to choose dose levels that would adequately span the range of fetal and maternal lethality [1, 2, 4, 5, 14, 17, 18, 27, 30]. As a vehicle and handling control, one group of animals was given a maltose-dextrin (MD) solution (PO). The MD solution was isocaloric to and pair-fed with the 9000 mg/kg/day alcohol dose.

Pregnant females in the nine alcohol-plus-cocaine groups were given doses of this drug combination in a fixed-ratio of 100:1 in accordance with animal weight. Specifically, the combination drug groups received either 1000 + 10, 2000 + 20, 3000 + 30, 4000 + 40, 5000 + 50, 6000 + 60, 7000 + 70, 8000 + 80 or 9000 + 90 mg/kg/day of ethanol + cocaine HC1. As a vehicle and handling control group, one group of animals was paired to the 9000 + 90 mg/kg/day alcohol- plus-cocaine group. The animals in this control group received the appropriate combination of saline (SC) and MD

(PO) vehicle solutions. The 100:1 fixed ratio was chosen because it approximately equated the two drugs in terms of reproductive toxicity. For example, the maternal median lethal dose (LD.~0), using our dosing regimen, proved to be 8100 mg/kg/day [18] and 74 mg/kg/day [15] for ethanol and cocaine, respectively. The fetal LDso points of 6300 mg/kg/day ethanol [18] and 71 mg/kg/day cocaine HCI [15] also approximated a 100:1 ratio.

Initially, 5 to 7 dams were assigned to each treatment group. Some animals eventually proved not to be pregnant, lowering most group sizes, to n =4 or 5. An additional control group of nontreated, ad lib fed and watered animals (n= 11) was employed for comparison purposes. Pair-feeding tech- niques were not employed because the present study focused on dose-response relationships, not mechanistic questions such as the contribution of undernutrition. Moreover, the higher dose groups were expected to have high mortality rates, making it difficult to pair-feed control groups to them in a meaningful way.

Each daily dose of drug (or vehicle) was split evenly with the first portion administered between 9-10 a.m. and the second portion administered between 3-4 p.m. To obviate problems associated with skin ulcerations, cocaine solution volumes greater than 0.30 ml were injected into multiple sites. While some skin ulcerations did occur in the higher dose groups (60 mg/kg and above), these were treated with antibiotic ointment and healed rapidly. We avoided injecting the cocaine solution in the nape of the neck since this region seemed unusually sensitive to skin ulcerations [15]. We also systematically varied the injection sites along the animal 's back and sides to avoid injecting near recently injected, and possibly inflamed, areas.

All animals had free access to food (Teklad rat pregnancy diet) and water. Daily records were maintained on maternal weights and food/water consumption. The pregnant (and nonpregnant) dams were sacrificed by carbon dioxide inha- lation on presumed gestation day 20 ( te rm=day 21). The fetuses (if any) were removed from the uterine horns, counted and weighed. The uteri were subsequently placed in a solution of ammonium sulfide and saline (10% v/v). The next day, the stained uteri were placed between glass slides and examined for implantation sites.

This study examined several standard parameters that have proven to be useful measures of maternal and fetal toxicity (i.e., maternal and fetal mortality, maternal weight gain, maternal food/water consumption, fetal weight). The interactive effects of alcohol and cocaine on these variables were evaluated by the isobolographic procedure. While this study represents the first time this procedure has been applied to prenatal drug exposure, it has been used in other toxicology studies [9, 10, 16, 22, 24, 32]. The isobolographic procedure is reviewed in detail elsewhere [22]. Briefly, the isobologram is defined as lines connecting dose pairs which are equieffective in regard to an adequately selected endpoint. It is a powerful method of graphically determining whether drugs are synergistic (mutually potentiating), merely additive, infraadditive, noninteractive (operating independ- ently), or antagonistic with regards to a chosen endpoint [e.g., median lethal dose (LDs0)] which is derived through regression analysis. Linear addition is the simplest case. It would occur if the two drugs were pharmacologically identi- cal. The data for such an interaction would lie along a straight line between the points of equal effect for each drug alone. Data to the lower left would indicate synergism; data to the upper right would indicate infraaditivity. Data points

A L C O H O L AND COCAINE 357

g/kg

8 Q) of) 0

a - - 6 0

0 ¢.)

<~ 4

a 2

LD 50 for Maternal Mortality ." °.- °.-

.o. . "

o° °.+°°°

°+°°°" °°°°

°°°°°°°°

°°°°°°°"

20 40 60 80 mg/kg

Daily Cocaine Dose

FIG. 1. Isobologram for the interaction of alcohol and cocaine treatment with respect to Maternal Mortality. The LDs0 points for each drug are plotted with their 95% confidence limits. The expected locations of LDs0 points for alcohol and cocaine combinations, given linearly additive effects, are depicted by the solid diagonal and associated confidence interval (shaded region). The dotted line going through the origin gives the loci of fixed-ratio (100:1) treatments of alcohol and cocaine. If the LDs0 for the alcohol and cocaine combination is below the diagonal, this would indicate synergism (i.e., more than additive). If the LDso for the drug combination is above the diagonal (as shown here), this would indicate infraadditivity. If the LD~0 for the drug combination lies along the rectangle formed by the origin and the two single-drug LD~0 points, this would indicate no interaction. Data falling outside this rectangle indicate antagonism.

along the rectangle formed by the origin and the two single- drug endpoints (e.g., LD~0 points) indicate no interaction, and data beyond this rectangle indicate drug antagonism (see Fig. 1).

To apply the isobolographic procedure, dose-response curves for all selected endpoints (e.g., maternal LD,~0) were constructed for each drug alone and for the 100:1 alcohol- plus-cocaine combination. The maternal LD~0 points (for example), with their 95% confidence intervals, were estimated by probit analysis for each dose-response curve. These values were then plotted as isobolograms.

RESULTS

Maternal Mortality

The data for maternal mortali ty are displayed in Fig. 1. For the parameter of mortality, the LDs0 point was selected as the appropriate endpoint. The LD~0 point for cocaine and its 95% confidence limits are marked on the ordinate. The procedure was repeated for alcohol with the appropriate data marked on the abscissa. The LDs0 and 95% confidence limits for the 100: I fixed-ratio combination of alcohol-plus-cocaine are plotted on a dotted line passing through the origin. This line represents the locus of all possible 100:1 alcohol-plus- cocaine combinations.

It can be seen from Fig. 1 that the LDs0 point for alcohol and cocaine, when combined in a 100:1 ratio, is significantly displaced above the diagonal line joining the LD~0 points of

g/kg

8

0) 0

a - - 6 C) ¢.- 0 ¢..)

'~ 4

121 2

ED 50 for Reduced Weight Gain

20 4 0 60 80 rng/kg

Daily Cocaine Dose

FIG. 2. Isobologram for the interaction of alcohol and cocaine treatments with respect to Maternal Weight Gain, using the median effective dose (ED.~o) for a 50% reduction in expected weight gain as the endpoint. The data indicate a linearly additive effect for these two drugs with regards to the impairment of maternal weight gain.

the two drugs when given separately. This indicates that the combination of alcohol and cocaine in a 100:1 ratio has an infraadditive effect on maternal mortality. If this drug combination had an additive effect on the maternal LDs0, the LDso point for the drug combination would be placed on or about the diagonal joining the LD~0 points of the individual drugs. If the drug combination had a synergistic effect, the LDs0 point would be significantly displaced below the diagonal. If the drug combination had a noninteractive or antagonistic effect, respectively, the LDs0 point for the drug combination would be at a right angle to the LDs0 point of an individual drug or significantly displaced beyond the rectangular region formed by joining the individual drug LD.~0 points at right angles.

Maternal Weight Gain

The data for maternal weight gain during treatment are displayed in Fig. 2. For this parameter, the median effective dose (EDs0) for a 50% reduction in expected maternal weight gain was selected as the endpoint. The criterion of a 50% reduction in expected maternal weight gain was selected because this was the approximate amount of weight reduction reported in previous developmental toxicology studies using maximum alcohol dose levels of 7000 mg/kg/day [4,5]. Moreover, the use of more extreme levels of weight gain reduction was compromised by the fact that too few of the surviving dams showed reductions greater than 50%.

The expected maternal weight gain during treatment (i.e., the difference between gestation day 20 and day 7 maternal weights) was based on the average weight gains of the nontreated and 3 vehicle control groups. Each group was weighted equally (i.e., N=4) with group datum (rather than the weight gain for individual dams) as the unit of measure. The data from the 4 control groups did not differ significantly, F(3,19)=2.389, p>0.10, allowing these means to be averaged to provide a single measure of expected maternal

358 CHURCH, DINTCHEFF AND GESSNER

g/kg8 t ED 50 for Reduced Food Consumption

0 3 0

a - - 6 0 t - O 0

a 2

20 40 60 80 mg/kg Daily Cocaine Dose

FIG. 3. Isobologram for the interaction of alcohol and cocaine treatments with respect to Maternal Food Consumption using the ED~0 for a 20% reduction in normal food consumption as the endpoint. The data suggest a linearly additive effect for these two drugs with regards to the reduction in maternal food consumption during pregnancy.

weight gain. This value equaled 93.4 g. Thus, a maternal weight gain of 46.7 g represented a 50% reduction in the expected value. Each female in each group was then nomi- nally categorized as falling (a) above or (b) equal to or below this point. These dichotomized data were then analyzed by probits, one probit for each drug condition (i.e., alcohol, cocaine, alcohol-plus-cocaine).

The data in Fig. 2 shows that the interactive effect of alcohol-plus-cocaine was linearly additive with respect to the maternal weight gain parameter.

Maternal Food~Water Consumption

The data for daily maternal food consumption during treatment are displayed in Fig. 3. For this parameter, the ED,~0 for a 20% reduction in expected daily food consumption was selected as the endpoint. The criterion of a 20% reduction was selected so it would parallel the criteria used for reduced fetal weight (see below). Moreover, reductions in food consumption much greater than 20% were not amenable to probit analysis because too few of the surviving dams showed reductions greater than 20%. The data in Fig. 3 show that the interactive effect of the alcohol-plus-cocaine combination was linearly additive with respect to the maternal food consumption parameter.

Daily maternal water consumption was adjusted to include fluid introduced by oral or subcutaneous injections, There were significant dose-dependent decreases in expected dally maternal water consumption in the cocaine- only, F(7,41)=7.227, p<0.001, alcohol-only, F(7,35)=26.424, p<0.001, and alcohol-plus-cocaine groups, F(8,39)=5.870, p<0.001. The EDso points for a 20% reduction in daily water consumption for the cocaine, alcohol, and cocaine- plus-alcohol were estimated by probit analyses to be 100.1 mg/kg/day cocaine HC1, 6700 mg/kg/day ethanol, and 5080

g/kg

8 ~D

0 a -- 6 0

0 0

. _

2 °°°

°°°°°° °,°,°

.°°°° °°°

LD 50 for Fetal Mortality ,•*

..•**** ..-

20 40 60 80 mg/kg Daily Cocaine Dose

FIG. 4. Isobologram for the interaction of alcohol and cocaine treatments with respect to Embryonal + Fetal Mortality, using the ID~o points as the endpoint. The data indicate an infraadditive effect for these two drugs with respect to fetal mortality.

+ 50.8 mg/kg/day alcohol-plus-cocaine. Since the ED~o point for the cocaine-only condition was beyond the highest dose given (i.e., 100.1 versus 90 mg/kg/day), no meaningful confidence intervals could be generated by the probit analysis. Nonetheless, inspection of the above EDs0 points strongly suggests that the interactive effect of alcohol and cocaine on water consumption tended to be infraadditive, or possibly additive. That is, if graphed, the EDs0 point for the drug combination would be positioned modestly above the diagonal line connecting the ED,~0 points of the individual drugs.

Conceptus Mortality

The data for conceptus mortality (embryonal + fetal losses) are displayed in Fig. 4. Conceptus mortality was cal- culated on a group basis. It can be seen from Fig. 4 that the interactive effect of the alcohol and cocaine combination on the conceptus LD~0 was marginally infraadditive.

Fetal Weight

The data for fetal weight, as per gestational day 20, are displayed in Fig. 5. For this parameter, the EDs0 point for a 20% reduction in the expected average weight for any given litter was selected as the endpoint. The criterion of a 20% reduction in expected average fetal weight was selected because of its use in the Collaborative Behavioral Teratology Study [7]. Moreover, the use of more extreme levels of weight reduction was compromised by the fact that too few of the full-term fetuses showed reductions greater than 20%.

Fetal weight data were handled in the same manner as described for maternal weight gain, except the litter datum was used as the unit of measure. The expected fetal weight was based on the average fetal weights of the nontreated and the 3 vehicle control groups. The data from the 4 control groups did not differ significantly from each other, F(3,19) = 1.698, p >0.10, allowing their data to be averaged to

A L C O H O L AND COCAINE 359

cO O

- 6

0

<~

c~ c~

g/kg ED 50 for Reduced Fetal Weight

/ /

/ /

/ /

/

20 40 60 80 rng/kg

Daily Cocaine Dose

FIG. 5. Isobologram for the interaction of alcohol and cocaine treatments with respect to Fetal Weights on gestation day 20, using the ED~ for a 20% reduction in expected fetal weight as the endpoint. The data indicate a linearly additive effect for these two drugs with respect to the reduction in fetal weight. Deficiencies in the data distribution prevented the probit analyses from generating the 95% confidence intervals.

provide a single measure of the expected average fetal weight for a given litter. This value equalled 3.72 g. An average fetal weight value of 2.97 g for any given litter, thereby, represented a 20% reduction in the expected value. The data in Fig. 5 suggest that the interactive effect of alcohol-and-cocaine was additive with respect to decreased fetal weight.

DISCUSSION

Previous drug interaction studies in developmental toxicology have relied solely on dose-response curves and analyses of variance for statistical evaluation (e.g., [3,41]). This makes it impossible to determine if the level of interaction is synergistic, additive, or merely infraadditive. The use of the fixed-ratio method of the isobolographic procedure permitted us to determine the level of interaction between alcohol and cocaine on pregnancy outcome in the Long- Evans rat. The level of drug interaction was dependent on the pregnancy variable being studied. That is, the alcohol- and-cocaine combination had linearly additive effects on reduced maternal weight gain, reduced fetal weight, and reduced maternal food consumption; while infraadditive effects were observed for maternal and fetal mortality, and reduced maternal water consumption. While there were no synergistic effects observed, it is important to realize that even linearly additive and infraadditive effects indicate that the drug combination is more toxic than the use of either drug alone. By constrast , if any two drugs have nonadditive or antagonistic effects, the levels of toxicity are unchanged or reduced, respectively [9, 10, 16, 22, 24, 32]. In extrapolat- ing to the human situation, therefore, these results of additive and infraadditive effects suggest that the combined use

of alcohol and cocaine by pregnant women may pose a greater risk to maternal and fetal health than the use of either drug alone.

When designing the present study, we speculated that alcohol and cocaine would have additive or synergistic effect. Instead, the interactive effects of these two drugs on pregnancy variables proved to be additive and infraadditive, depending on the pregnancy variable in question. Isobolog- rams falling in the infraadditive region pose the question whether the apparent infraadditivity is due to the agents act- ing by different mechanisms or whether other explanations should be sought [22]. For example, alcohol alters the distribution of cocaine, increasing the brain-to-plasma cocaine concentration ratio [38]. This may be a mechanism for the increased maternal and fetal/embryonal toxicity. On the other hand, all maternal deaths in the alcohol-plus-cocaine groups occurred within the first 3 days after treatment onset. It would appear that this effect, in the main, was due to action of alcohol since maternal deaths in the alcohol-only groups occurred early in treatment [18], while maternal deaths in the cocaine-only groups mostly occurred late in treatment [15].

The interactive effects on maternal weight gain and food consumption were linearly additive. Usually, this would suggest that the drugs had a similar joint action on these variables [22]. It is conceivable, however, that the two drugs had an independent joint action (i.e., producing the same effect by entirely different mechanisms). For example, reduced maternal weight gain and food consumption in the alcohol-only dams were most likely due to alcohol 's caloric content (making animals feel satiated) and alcohol 's sedation (making animals incapable of eating). In the cocaine-only dams, on the other hand, appetite suppression through the stimulation of dopaminergic mechanisms and the excessive burning of calories through increased motor activity were likely casues in the reduced maternal weight gain and food consumption.

In addition to the above pregnancy outcome variables, it should be mentioned that prenatal alcohol exposure [18] and prenatal cocaine exposure [15] can result in placental abruption, fetal edema, cephalic hemorrhages, and physical malformations. While placental abruption, edema, and hemorrhages were observed in a number of fetuses prenatally exposed to alcohol-plus-cocaine, there was no strong evidence of a drug interaction on these variables. Moreover, there were no incidences of physical malformations in any of the alcohol-plus-cocaine fetuses, as assessed by gross external examination. By constrast, we have observed instances of physical anomalies in Long-Evans rat fetuses exposed to alcohol [18] and cocaine [15]. The incidence rates of these anomalies were small (less than 3%), however. Conse- quently, it is uncertain if the alcohol-and-cocaine combination had a protective (i.e., antagonistic) effect on the physical deformities that are caused by these two drugs when given separately to the Long-Evans rat. It is also conceivable that the higher mortality in the alcohol-plus-cocaine group involved deformed concepti , lowering the sample size of deformed fetuses in these treatment groups.

It is worth noting that there were considerable decreases in maternal weight gain and food consumption during pregnancy by dams in all the drug groups. These effects may be important with respect to the high degree of mortality. That is, undernutrition itself may have interacted with drug toxicity to enhance fetal and maternal mortality. The time during pregnancy for drug treatment can also have a significant in-

360 C H U R C H , D I N T C H E F F A N D G E S S N E R

fluence on pregnancy outcome. These issues are discussed in more detail e l sewhere [3,15].

In the present study, analyses focused on select endpoints. Multiple i sobolograms showing the LD20, LDs0 and LD80 points (for example) can yield additional informa- tion [9]. Proper applicat ion o f multiple i sobolograms would require , however , a larger animal populat ion than used in the present exper iment . Our data analyses also focused on a select set of maternal and fetal variables and employed only one fixed-ratio combinat ion o f a lcohol and cocaine. It is conceivable that the nature of a lcohol and coca ine ' s interact ion may vary as a funct ion of the f ixed-rat io chosen jus t as it seems to vary as a funct ion of the maternal and fetal variables studied. That is, one drug can both antagonize and enhance the effects of a second drug depending on the parameter and endpoint recorded and the part icular drug combinat ion adminis tered [16]. The use of multiple f ixed-ratio drug combinat ions , the histological examinat ion of internal organs, and multiple i sobolograms represent the next steps in our research.

One t roub lesome issue we encounte red in applying the isobolographic procedure to the study of reproduct ive toxicology concerned the select ion and just i f icat ion of adequate endpoints . For example , we selected a 20% reduction in expec ted fetal weight gain as one index of fetal toxicity. While our select ion o f this endpoint was not arbitrary, future research may establish a 10% reduct ion (for example) as a more appropr ia te index of fetal toxicity. We wish to emphas ize that the various endpoints employed by the present s tudy are not to be regarded as thresholds or hal lmarks for biologically significant effects. Instead, they were chosen, in part, because they represented relat ively un- equivocal levels of drug-induced toxici ty and could serve to illustrate how the isobolographic procedure can be applied to the study of reproduct ive toxicology.

In conclusion, it is our feeling that the isobolographic procedure offers researchers in reproduct ive toxicology a meri tor ious method to study drug interactions. We say this for several reasons. First , the procedure necess i ta tes the use o f a broader dose range and more dose levels than tradi- t ionally used by other methods. The result is a c learer and more comple te picture of a drug 's dose-dependent effects and a more comple te character iza t ion of the interact ive effects o f two or more drugs. Second, the procedure is more sys temat ic and theoret ical ly sound in that it requires the equat ing of two or more drugs on adequate endpoints . Third, it is powerful and informat ive in that it can readily distin- guish be tween 5 different levels of drug interact ion (i.e., synergism, additivity, infraadditivity, nonaddit ivi ty, and antagonism). This lat ter at tr ibute is part icularly important because the l i terature on drug interactions is muddled by numerous reports that certain drugs have synergistic effects when in fact the data, if graphed according to the isobolographic procedure , show merely addit ive or infraaddit ive effects [9, 16, 32]. In o ther words, most researchers mistak- ingly interpret any enhancement of one drug 's toxici ty by a second drug as ev idence of synergism, not realizing that enhanced toxici ty can occur at three distinct levels (i.e., synergism, additivity, and infraadditivity).

ACKNOWLEDGEMENTS

We thank K. Schmid for the illustrations and K. Callanan and N. Sarhan for manuscript preparation. This project was supported in part by BRSG S07 05938-02 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health and by NIDA grant DA05536 awarded to Dr. Church.

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the interactive effects of alcohol and cocaine on maternal and fetal toxicity in the long-evans rat

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