In vitro developmental toxicity of five direct-acting alkylating agents in rodent embryos: Structure-activity patterns

TERATOLOGY 40:199-210 (1989)

In Vitro Developmental Toxicity of Five Direct-Acting Alkylating Agents in Rodent Embryos: Structure-Activity Patterns

ELAINE M FAUSTMAN, ZAMYAT KIRBY, DANIEL GAGE, A N D MICHAEL VARNUM Department of Eniitronmental Health, SC-34 tE M F , 2 K , D G , M V ) and Child Deueloprnunf and Mental Retardation Center f E M F I . University of Washington, Seattle, Washington 98195

A RSTRACT Five direct-acting alkylating agents were examined qualitatively and quantitatively for their ability to produce developmental toxicity in rodent postimplantation embryos. These agents were structurally related and were capable of donating either a methyl (methylnitrosourea, MNU; methylnitronitrosoguanidine, MNNG; methyl methanesulfonate, MMS) or ethyl (ethylnitrosourea, ENU; ethyl methanesulfonate, EMS) group to nucleophiles. These agents' reactivities were known to differ. In day 10 rat embryos in vitro a single, 2-hour exposure was shown to be sufficient to elicit dose-dependent increases in embryo lethality and malformations. Qualita- tively, the patterns of embryo malformations reported in treated embryos paralleled those observed in in vivo studies, especially in regard to adverse effects on central nervous system and craniofacial systems. Quantitatively, the order of potency of these agents in vitro was: MNNG > MNU > ENU > MMS > EMS. In vivo studies reported a different order of potency. In vitro, methylating agents were consistently more potent than ethylating agents. Other chemical properties such as nucleophilic reactivity or half-life under physiological conditions could not explain observed potency relationships. Future investigation of other chemical properties of these agents such as specific alkylation and carbamylation reactivities may expand these initial structure-activity observations.

Exposure to alkylating agents occurs daily through air. water. and food as this class of chemicals has representatives in most groups of environmental and occupational pollutants (Fine, '78; Mirvish, '77; Preussmann, '84; Lijinsky, '86). Because of the widespread human exposure to these agents, we were interested in their potential developmental toxicity. Although, as a group, alkylating agents are defined by their ability to donate alkyl groups to nucleophiles, these agents differ widely in their chemical reactivities (Manson, '81; Colvin, '82). We hypothesized that if such compounds proved to have differential developmental toxicity that correlated with their chemical properties, then possible mechanisms of their teratogenic action might be suggested.

The developmental toxicity of five model alkylating agents was examined using the rodent postimplantation culture system. These agents differ in chemical properties in two main aspects. First, they donate either a methyl (methylnitrosourea, MNU; methylnitronitrosoguanidine, MNNG; or methyl methanesulfonate, MMS) or ethyl (ethylnitrosourea, ENU; or ethyl methanesulfonate, EMS) group. Their chemical reactivities also differ with respect to their dependence or independence on the chemical nature of the nucleophilic site that they

Received Ju ly 8. 1988: accepted February 3. 1989. A preliminary report ofthis work was presented at the Annual

Meeting of the Society of'roxicology. Sail Diego, CA. March 1985 ! T / I P Toxicologist 5 : 138, 19851. This research \*'as supported by NIH grant ES-03157.

6 1989 ALAN R. LISS. INC.

200 E.M. FAUSTMAN ET AL.

TABLE 1. Deuelowmental toxicitv effect levels for five model alkvlating agents Nucleophilic LCso ('2.1.) MC,o (C.1.) PC33 (C.1.) DC33 ('2.1.)

Agent S1 mechanism' (LLM)~ (uMP ( u M ) ~ , ~ (uM)~,, MNNG 0.42 SN1 4.2 (3.9-4.5) 1.8 (1.6-2.1) 1.1 (0.9-1.3) 1.8 (1.4-2.5) MNIJ 0.42 SN1 80 (62-116) 33 (24-401 38 (14-104) 52 (29-921 . ~~ .~ ~ ~. ~, ~~.

ENU 0.26 SN1 159 (143-184) 70 i56-82j 109 (61-194) a6 (64-116) MMS 0.83 SN2 637 (585-692) 300 (259-333) 320 (244-419) 350 (312-392) EMS 0.64 SN2 (SN1) 7,517 (6,450-9,135) 4,266 (3.204-5.863) 3.000 (1.176-7,650) 4,600 (3,170-6.670)

'Swaine-Scott S factor is a comparison of the chemical reactivity of each agent to the reactivity of the hydroxide ion (Bartsch et al.,

'SN1 = nucleophilx substitution, 1st order; SN2 = nucleophilic substitution, 2nd order. 'C.I. = 95% confidence interval. 4PC33 = the concentration that produces a 33% decrease in embryo protein content. 5DC33 = the concentration that produces a 33% decrease in embryo DNA content.

'82).

will attack (Ingold, '69). Agents such as MNNG, MNU, and ENU are classified as SN1 type agents and react through nucleophilic substitutions to form an intermediate (alkylcarbonium ion, rate-limiting step), which rapidly interacts with nucleophiles to produce alkylation products. These reactions are first order with respect to the concentration of the original alkylating agent and are independent of the substrate concentration. In contrast, SN2 alkylation reactions are dependent both on the nucleo- philicity of the site and on the concentration of the alkylating agent (Bartsch et al., '82; Manson, '81; Lawley, '74, '76). MMS and EMS are examples of SN2-type agents, although EMS has also been suggested to have some SN1-type characteristics (Bartsch et al., '82). The Swain-Scott S factor was developed to enable comparisons of the chemical reactivity of various alkylating agents and compares each agent's reactivity to the reactivity of the hydroxide ion (Swain and Scott, '53). Table 1 shows the nucleophilic mechanism and S reactivity factor for each of the five alkylating agents tested.

The carcinogenic and mutagenic properties of these agents have been extensively investigated using both in vivo and in vitro systems (Singer, '75; Lijinsky, '86). How- ever, much less attention has been given to their developmental toxicity. It is known that all five agents do cause developmental toxicity in in vivo rodent studies, and ENU was chosen as one of 47 candidate compounds for in vitro teratogenicity test validation (Manson, '81; Sweet, '87; Smith et al., '83; Bochert et al., '78a,b). The objective of our study was to examine five agents with definable chemical properties in an initial effort to characterize qualitatively and quantitatively the in vitro developmental

toxicity of these agents and identify common chemical properties that would merit further investigation as possible contribu- tors to these agents' developmental toxicity.

MATERIALS AND METHODS Chemicals

ENU, EMS, MMS, and MNNG were pur- chased from Sigma (St. Louis); MNU was from K & K Biochemicals (Plainview, NY). Because of the lability of several of these chemicals, all chemicals were weighed or dispensed and dissolved immediately prior to use; they were added to culture bottles without delay after gassing was complete, and the embryos were added. The pH of all solutions was also carefully monitored. The chemicals were dissolved in the following solutions: MNU in 11 mM sodium citrate buffer, pH 6.0; ENU in 0.1 M sodium acetate buffer, pH 6.5; MMS in saline; EMS in Hank's Balanced Salt Solution (HBSS), pH 7.4 (Gibco, Grand Island, NY); and MNNG in absolute ethanol. When ethanol was used as a solvent it was evaporated under a stream of air prior to addition of culture media. MNU and ENU are shipped containing a stabilizer (acetic acid), which contrib- utes substantially to their weight; all concentrations shown here are corrected for this factor. Stock solutions and culture medium were chemically inactivated before disposal (saturated NaOH was used for ENU and MNU; 10% sodium thiosulfate for EMS and MMS; 3.3% sulfamic acid in 6 N HC1 for MNNG) (Castegnaro et al., '83; Montesano et al., '79).

Animals Primigravida Sprague-Dawley (Wistar-

derived) rats were obtained from Tyler Lab- oratories (Bellevue, WA). Pregnant animals were received between days 4 and 7 of

IN VITKO TERATOGENICITY OF ALKYLATING AGENTS 20 1

pregnancy and were kept in plastic cages on crushed corncob bedding material (Ander- sons, Delphi, IN). They were housed in the animal facility of the Department of Envi- ronmental Health where they had access to food (Wayne Research, Chicago, IL) and water ad libitum. A 14-hr light, 10-hr dark lighting schedule was maintained. The morning following copulation was desig- nated day 0 of gestation.

Embryo culture The animals were anesthetized with

ether a t 0900 hr on the 11th day of pregnancy !day 19 c~hrycs! , nnd hloo? waq collected from the abdominal aorta. The pooled blood was centrifuged immediately at 5"C, and the red cells and fibrin clots were discarded. The uteri were removed to a dish of cold, sterile HBSS, and the individual implantation sites were removed. Embryos were explanted by a modification of methods described by New ('78). Decidua, trophoblast remnants, pari- etal yolk sac, and Reichert's membrane were carefully removed, leaving the vis- ceral yolk sac, ectoplacental cone region, and amnion intact. Embryos having 10+2 somites were selected for culture. Embryos from each litter were distributed evenly among bottles containing 14 ml of HBSS, pH 7.4, which had been gassed for 20 min with 5% C 0 2 in air and heated to 37°C. Test chemicals were added, and bottles were capped and incubated for 2 hr a t 37°C with constant rotation (20-25 rpm). Following this, embryos were removed, rinsed 2 x in warmed (37°C) HBSS, and placed in bottles containing prygassed ( 5 7 . CO, in air), heated, complete culture medium (Faust- man-Watts et al., '83). The rest of the culture procedure was as described in that report.

Assays After 24 hr in culture, viable embryos

(with heartbeat and active yolk sac circula- tion) were examined under a dissecting mi- croscope as previously described (Faust- man-Watts et al., '83). All embryos were assessed without knowledge of treatment conditions. Measurements of head length, crown-rump length, somite number, and limb bud development were obtained. Mal- formations and growth indices were re- corded, and representative embryos were photographed and fixed in glutaraldehyde

(2.5% in 0.1 M sodium phosphate buffer, pH 7.4) for histological examinations. They were then dehydrated, embedded in hy- droxyethyl methacrylate, sectioned with glass knives a t 2 to 3 pm, and stained with toluidine blue. An embryo was considered to be abnormal if it had one or more grossly observable malformations. All malformation types were examined histologically for evidence of abnormality.

Protein and DNA determinations were made on embryos ultrasonically disrupted in 0.1 M sodium phosphate buffer (pH 7.4). Protein concentration was determined ac- cording to the method o f Bradford ('76). DNA content was assessed fluorometrically (Labarca and Paigen, '80).

Statistics One-way analysis of variance (ANOVA)

was used to determine the statistical significance of differences between treated and control samples (Number Cruncher 2.1, Dr. Jerry L. Hintz, Kaysville, UT). Ordered contingency tables were used to partition the overall chi-square statistic to discern ordered trends and sources of variation (Everitt, '77). PC3:$ and DC3:3 calculations and parallelism tests were conducted using the methods of Litchfield and Wilcoxon ('49). PC33 and DC33 are the concentrations of a compound that reduced the protein content (P) or DNA content (D) by 33% of the concurrent controls. The significance level chosen was P < .05. Numbers of embryos evaluated are given in the tables and figure legends. LCrjo and MC5,, calculations were performed using a computer program dcvcloped by H.R. Lieberman ('83) hased nn the methods of' Finney ('78). LCrjo and MCSo values are the concentrations of a compound that increased lethality (L) and malformations (M) to 504.

RESULTS

Figure 1 shows the log dose-lethality curves for five model alkylating agents. All five of the agents tested produced significant dose-dependent increases in embryo lethality. The five alkylating agents produced these effects over a fourfold range in concentration. The order of potency determined in this culture system is MNNG > MNU > ENU > MMS > EMS. When the log dose-response curves for embryo lethality were compared using the method of Litch- field and Wilcoxon ('49), the curves for

202 E.M. FAUSTMAN ET AL

I 10 I00 lpoo l0,Ooo

Log Concentration ( p M )

Fig. 1. Log dose-response data for embryo lethality plotted for MNNG (0 ) . MNU [m), ENU (A), MMS to), and EMS (U) exposed embryos. Each curve represents averaged data for 143-212 embryos from a minimum of 15 different rats and three independent experiments.

I I L L L . L L L _ . . . . . . . I . , . 10 '0C OW 10000

Log Concentrotion ( p M )

Fig. 2 Log dose-response data for embryo malformations for MNNG (*), MNU (D), ENU (A), MMS (o), and EMS (0) exposed embryos. Each curve represents averaged data for 143-312 embryos from a minimum of 15 different rats and three independent experiments

MNU, ENU, MMS, and EMS were parallel; however, the hypothesis of parallelism was rejected for MNNG (P > .05).

Figure 2 shows the log dose-response curves for malformations produced by these alkylating agents. Significant dose-dependent increases in malformations were produced by each of these direct-acting compounds. These agents elicited malformations in the whole embryo culture system in the same order of potency as was seen for their embryo lethality effects. However, the

dose-response curves for malformation were shifted to the left of the dose-response curves for lethality for each agent. MNNG was approximately four orders of magnitude more potent than EMS in eliciting embryo malformations. Table 1 shows the LCs0 and MCso values (and 95% confidence intervals) for these agents. Tests for parallelism re- vealed that the log dose-response curves for MNU, ENU, MMS, and EMS are consistent with a hypothesis of parallelism. The curve for MNNG-induced malformations was also

IN VITRO TERATOGENICITY OF ALKYLATING AGENTS 203

parallel to all other curves except the EMS malformation curve.

The ratios of MC50/LC50 values for these alkylating agents were 0.43, 0.41, 0.44, 0.47, and 0.57 for MNNG, MNU, ENU, MMS, and EMS, respectively. All of these ratios were less than 1, indicating that effects on embryo malformations were observed a t lower concentrations than were effects on embryo viability. This same pattern of toxicity was seen a t low doses when the MClo/LClo ratios were examined.

Figure 3 shows photographs of representative embryos exposed to the alkylating agents testcd in this study. .4!! 3.ffccted embryos had a reduction in prosencephalon size relative to crown-rump. In addition to hypoplasia of the prosencephalon, treated embryos exhibited cephalic edema, prima- rily in the rhombencephalon (Fig. 3a); debris in amniotic cavities and neural tubes (Fig. 3c); and micro- and anophthalmia (Fig. 3a,b,e). Abnormalities of flexure resulted from adhesions of the neural tube a t the level of the limb to the neural tube a t the level of the optic cup (Fig. 3a) and incom- plete rotation of the tail with a kink in neural tube a t the level of the limb (Fig. 3b,d,e). Other neural tube abnormalities were also observed (Fig. 3). As well, abnormalities of the optic cup, neural tube, and flexure occurred in a dose-dependent man- ner with respect to incidence and severity.

Figure 4 shows representative histological sections through the cephalic region and optic cups of control and treated embryos. Figure 4a,b shows a section of a control embryo at low and high magnification, respectively. At 370 7 , the iiornial, well-pre- served epithelium of this control embryo can be contrasted with the necrotic neural epithelium observed in embryos exposed to 75 pM MNU (Fig. 4d), 500 pM MMS (Fig. 4f), 4 pM MNNG (Fig. 4h), 120 FM ENU (Fig. 4j), and 4 mM EMS (Fig. 41). Even when specific neural tube abnormalities were absent, histological examination re- vealed that all embryos that were mal- formed after exposure to these alkylating agents displayed necrotic cells and debris in the neural tube region.

Table 2 shows the effects of alkylating agents on embryo growth parameters. Us- ing one-way ANOVA, significant dose-dependent decreases in protein, DNA, crown- rump length, and somites were observed with all agents tested. Minimal changes in

limb size were noted (data not shown). Table 1 gives concentrations of these agents that produced a 33% decrease in protein and DNA content. A 33% decrease was chosen because a decrease in macromolecular content of 250% is not usually compatible with viability. The potency of the alkylating agents to produce decreases in these growth parameters was the same as their relative potency to elicit embryo lethality and malformations.

DISCUSSION

In this study five direct-acting alkylating sger?ts w e r ~ euaminrd qi ial i tat iwly and quantitatively for their ability to produce developmental toxicity in vitro. These agents were structurally related and were capable of donating either a methyl or ethyl group to nucleophiles. However, their reactivities were known to differ dramatically. These agents were selected not only on the basis of their structure-activity relationships but also because they were model compounds known to produce developmental toxicity in vivo and were direct-acting so that differences in metabolic activation reactions did not complicate our comparisons.

In the whole embryo culture system these agents were capable of eliciting dose-dependent increases in embryo viability and increases in embryo malformations. In our culture system, the order of potency for these development toxicants was MNNG > MNU > ENU > MMS > EMS for all of the various endpoints examined. As indicated by the ratio of MC50/LC50 values for these agents, effects on embryo morphogenesis occurred at lower concentrntinns than ef fects on embryo viability. These observations were consistent with effects observed a t low doses (MClo/LC1o). Parallelism tests suggested that the dose-response curves for four of the agents are parallel for lethality and malformation. This is consistent with a hypothesis that these agents are working through a common mechanism to produce adverse developmental outcomes. The MNNG curve for embryo lethality was not parallel to the four other curves, but its curve for malformations was parallel to all other curves except EMS. These observations are consistent with the idea that MNNG induces malformations via similar mechanisms as the other agents (except EMS) but are inconsistent with a hypothesis for a common mechanism for lethality.

204 E.M. FALISTMAN ET AL

Fig. 3. a: Embryo on top is the control; embryos below were exposed (L to R) to 56, 56. 38. and 38 pM MNU. The first embryo on the left had a n abnormal flexure resulting from a n adhesion of its neural tube a t the level of the limb to its neural tube a t the level of the optic cup. Not visible from this view, the embryo's neural tube is open from the prosencephalon to the rhombencephalon. It also has a severely reduced prosencephalon. The next embryo has edema of the cephalic regions and a reduced prosencephalon. The third has an open neural tube only in the prosencephalon and a reduced optic cup and prosencephalon. The fourth has edema in the cephalic regions, most notice- able in the rhombencephalon and a reduced prosencephalon ( w 61). b: Embryo on the left is the control; other two were each exposed to 375 pM MMS. Both have open neural tubes, reduced or absent optic cups. and reduced prosencephalons. The embryo on the right has an abnormal flexure (not fully rotated with a kink in the neural tube at the level of the limb) ( s 981. c: Embryos

on far right and left are controls; embryos in middle were each exposed to 120 pM ENU. They both display edema of the rhombencephalon and reduced prosencephalons; also they both had debris within their amniotic cavities and neural tubes, visible during dissection ( ~ 7 ' 7 ) . d: The embryo on the left is the control; the others were exposed cL to R) to 8,4. and 2 mM EMS. The embryos exposed to 8 and 4 mM EMS have abnormal flexures; the one exposed to 4 mM has a n open neural tube rostra1 from the prosencephalon-mesencephalon junction where there was a n amniotic adhesion. All three exposed embryos have reduced prosencephalons ( s 751. e: The embryo on t.he left is the control; the others were exposed ( L to R ) to 4, 3, and 2 pM MNNG. All three exposed embryos have abnormal flexures and reduced prosencephalons. dramatic in the 4 and 3 pM exposures. In addition, the embryo exposed to 4 pM has an open neural tube and a unilaterally missing optic cup ( x 89).


The patterns of malformations elicited by these agents were qualitatively similar and included striking effects on neural epithelium and hypoplasia of the prosencephalon. These agents also produced abnormally open neural tubes, micro- and anopthalmia, and flexure irregularities. These types of abnormalities are consistent with those observed in vivo, where these agents have been shown to produce in rodents a pattern of effects that includes eye, ear, craniofacial, and central nervous system defects (Sweet, '87; Manson, '81; Napalkov and Alexandrov, '68; Druckrey, '73; Wechsler, '73; von Krey- big, '65; Fox et al., '80; Givelber and DiPa- 010, '69; Diwan and Meier, '74; Ehrentraut et al., '69; Ivankovic, '79; Koyama et al., '70; Bochert et al., '78a,b, '81; Inouye and Mu- rakami, '78; Platzek et al., '82, '83, '87; Druckrey et al., '66). Not observed in our in vitro system, these agents have also been shown to produce musculoskeletal, cytolog- ical, and germ cell changes and body wall abnormalities in vivo. Because of the design of our 24 h r embryo culture system, our study was not able to identify these types of abnormalities. Consistent with our in vitro results were the observations of dose-dependent growth retardation and lethality in in vivo rodent tests.

In general, we have found the alkylating agents with SN1 nucleophilic substitution reaction characteristics and low Swain- Scott S values to be more potent as developmental toxicants in our in vitro culture system. However, these are generalizations based on limited numbers of test compounds where direct comparisons of S values to LC50 or MC50 values did not yield significant correlation coefficients. In addition, when two structurally related alkylating agents are compared, where the only differ- ence in structure is a methyl or ethyl sub- stituent, then the methylating derivative was always observed to be a more potent developmental toxicant (for example, MNU is more potent than ENU, and MMS is more potent than EMS).

In the case of other toxic endpoints of agents, mutagenicity and carcinogenicity, it is believed that the ability of these agents to alkylate selectively oxygen atoms such as O6 of guanine and O4 of thymidine most accurately reflects their biological activity (Loveless, '69; Frei et al., '78; Pegg and Nicoll, '76; Newbold et al., '80; Swenberg et al., '84). Limited in vivo studies suggest that

the O6 alkylations are important in determining the developmental toxicity of these agents (Platzek et al., '82, '83, '87; Bochert et al., '78a,b; Bochert, '75). These investiga- tors compared alkylation levels of embryonic DNA at equally effective teratogenic doses of three methylating agents, MMS (SN2) and MNU (SNI), and acetoxymethyl- methylnitrosamine, and one ethylating agent, EMS (SN2). A good correlation ex- isted between the DNA alkylation rate at the 06-guanine site in mouse embryos and the teratogenic potency (skeletal abnormalities) of the substances (Platzek et al., '87). However, these studies were plagued with low levels of alkylation and limited tissue availability. I t is not currently known in our system what the contribution of specific alkylation products is, but we are pursuing this question.

The direct-acting alkylating agents that were tested in this study are model compounds and are extremely reactive and un- stable under physiological conditions. Con- sequently, another factor that could determine potency is half-life (tIl2) at pH 7.4 at 37°C. Under these physiological conditions, the half-lives of these agents ranged from 0.34 h r for ENU to 11.6 h r for EMS (Bartsch et al., '82). When Lhe MCS0 or LCs0 values were compared to half-life, no statis- tically significant correlations were observed (r = 0.75 and 0.75, respectively, P > .05). Thus, half-life at physiological conditions did not appear to account for the differences in potency that we observed.

In vivo rodent studies have shown that these agents have LD5,, values that range from 90 mg/kg for MNNG (oral) to 350 mg/kg (i.p.) for EMS (Sweet, '87). The order of lethal potency in vivo following oral exposure is as follows: MNNG > MNU > MMS > ENU > EMS. This order of potency differed only in one respect from our in vitro developmental toxicity (MMS, ENU). The in vivo toxic dose low values for developmental toxicity (TDLo, the lowest dose given to any rodent species reported to produce any type of toxicity) can be compared. The following order of potency is observed: MNU > ENU > MMS > MNNG > EMS (EMS administered by parental route; others were a single i.p. dose). These TDL, values ranged from 1 mg/kg for MNU to 100 mg/kg for EMS (Sweet, '87). The relative ability of these agents to produce lethality or developmental toxicity in vivo


Figures 4a-h.


Fig. 4. Sections through the cephalic region and optic cups of control and treated embryos were photographed a t x 65 and x 270. Conditions were (a,b) un- treated control (HBSS and vehicle exposure only); (c,d) 75 pM MNU; (e,D 500 pM MMS; (g,h) 4 p M MNNG;

(ij) 120 pM ENU; and (k,l) 4 mM EMS. Sections from embryos treated with alkylating agents display cells and cellular debris sloughing into the neural tube and pycnotic nuclei. The section in k passes through the open neural tube of this embryo.

is not the same as the order of potency of these agents in vitro, probably hecause toxicokinct,ic factors play a critical role in determining in vivo values.

The in vitro results presented in this study can be compared to other in vitro studies. We previously (Solomon and Faust- man, '87) examined the effects of four alkylating agents on medaka fish embryos. The developmental toxicity of the agents examined was MNU > ENU > MMS > EMS, the same order we have observed in this study. However, approximately ten times the concentration used in the rodent embryo culture experiments was required to elicit developmental toxicity in medaka embryos. In vitro, these agents are also able to produce mutations. In the Ames assay the following order of mutagenic potential has been observed: MNNG > MNU > MMS > ENU > EMS. This order differs from our observa-

tions only in the ordering of ENU and MMS. Other reports have also suggested a lack of correlation between in vitro mutagenic and developmental toxicity potential (Faust- man-Watts et al., '84; Hales, '82).

In summary, these studies have demon- strated the in vitro developmental toxicity of five structurally related model alkylating agents in rodent postimplantation cultures, and their effects were qualitatively similar to in vivo rodent test results. Chemical properties such as nucleophilic reactivity or half-life could not explain observed potency relationships.

ACKNOWLEDGMENTS

Special thanks go to Elizabeth Walker, Steve Moss, and Jody Lottsfeld for technical assistance and to Azure Skye for typing the manuscript.

208 E.M. FAUSTMAN ET AI,

TABLE 2. Efficts of crlk\'lnting agents o n ernhr:vo growth pnmrrrcfers No. of Crown-rump Head

Concentration embryos Protein DNA length length Compound PM assessed (pg~embryor 1 pgiembryo) tmmr (min) Somi tes MNlJ 0

9.4 19 38 76

113 ENIJ 0

20

MMS

42 86

120 150 171 200

0 250 375 500 750

EMS 0 2,000 4,000 8.000

41 5

14 33 3 9 10 39

9 23 44 27 21 28 1 '1 4 0 34 3 2 39 37 51 33 40 42 41 13 28 14 18 29 26 27

165 + 69l 181 2 3 3 123 + 56 111 1 5 2 93 i- 45

83' 238 ' 72 247 I 4 6 245 - 30 175 I 87 159 + 56 114 L 19 100 t 46

145 + 49 1.50 1 79 89 48 81 * 29 57 1 102

141 - 54 108 L 38 81 - 45 63 2 31

128 - 47 168 ' 35 90 1 4 1 73 + 36 61 i 33 64 r 37 33 i 20

-

-

9.9 + 2.9 8.6 ! 2.5

11.4 + 4.7 7.5 t 3.9 5.1 L 1.8

,'i.32 9.2 f 3.6

11.0 1 2 . 3 8.1 f 2.8 6.2 i 2.9 4.6 + 2.0 2.9 2 1.2 5.9 '. 3.:3 I 4 06'

10 5 ' 2.0

6.8 - 1.6 5.8 2 1.7 3.7 I 0.9' 9.6 - 2.8 9.2 I 2.4 5.9 - 2.0 4.7 c 1.7

10.6 + 1.9 9.7 I 1.9 8.0 L 1.7 8.0 '. 2 0 7.1 2 2.3 5.2 I 1.2 5.3 c 1.4

10.4 . :% I

-

3.0 ! 0.2 3.0 2 0.1 2.9 t 0.2 2.8 '. 0.3 2.6 .i 0.2

*] 2' 2.8 + 0.4 2.8 I 0.3 2.9 f 0.4 2.6 1 0.3 2.6 0.3 2.6 . 0.1 2.3 i 0.2

.) I '$2

,'3 2 * 2 3.1 -+ 0 . 3 2.8 : 0.2 2.7 t 0.2

b) 4' 3.0 + 0.2 3.1 1 0 . 3 2.8 + 0.3

3.0 + 0.2 3.0 1 0 . 2 2.9 i 0.2 2.9 -* 0.3 2.9 = 0.3 2.7 i 0.3 2.6 2 0.2

2.32

-.

I.

I -

1.5 1 0 . 2 20.3 : 1.5 1.4 ?. 0.2 1.4 + 0.1 1.3 + 0.2 1.2 + 0.2

1.02 1.4 - 0.2 1.4 -+ 0.1 1.3 T 0.2 1.2 :. 0.2 1.2 + 0.3 1.1 :& 0.1 1.0 I 0.2

i . 8 ~ 1.6 ! 0.2 1 4 .r 0 3 1.2 ? 0.2 1.0 + 0.2 1.1 1 0 . 1 2 1.5 - 0.2 1.5 rt 0.1 1.2 - 0.3 1.1 2 0.2 1.5 + 0.1 1.5 ?. 0.1 1.4 i 0.1 1.4 - 0.2 1.3 i 0.2 1.3 + 0.2 1.2 I 0.2 -

19.4 + 1.3 20.7 -5 1.8 19.2 + 2.7 18.4 ..- 1.2

16.5' 19.7 L 1.5 20.8 2 2.1 20.1 t 2.4 19.0 + 2.1 19.4 -+ 1.6 18.4 I 1.7 16.5 t 2.8 15 7 + 1 2" 20.7 - 1.3 20.5 . 1 4 18.9 + 1.6 18.7 i 1.9 14.0 : 1.2' 20.2 -?. 1.5 20.0 I 1.9 18.5 - 1.7 18.2 I 1.7 20.3 -+ 1.2 20.9 I 1.5 19.6 I 1.5 19.9 + 1 6 19.0 c 2.0 18.1 ?- 1.9 17.1 c 2.0

14.5'

'Values are nieans % standard deviations. 'High rates of embryo lethality made assessments of these parameters very dif icult . .'Owing to ahnornialities of flexure, accurate crown-rump values could not he determined

LITERATURE CITED

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