Chem. Res. Toxicol. 1991, 4, 35-40
35
Effect on Mutagenicity of the Stepwise Removal of Hydroxyl Group and Chlorine Atoms from 3-Chloro-4- (dichloromethyl)-5-hydroxy-2( 5H)-furanone: ''C NMR Chemical Shifts as Determinants of Mutagenicity Robert T. LaLonde,*vt G a r y P. Cook,+Hannu Perakyla,? and Carlton W. Dencet Departments of Chemistry and of Paper Science and Engineering, College of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210 Received June 28, 1990
The response of mutagenicity to the stepwise replacement of chlorine atoms and the hydroxyl (MX, 1) was degroup by hydrogen in 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone termined in several assays by using Salmonella typhimurium tester strain (TA100). In all, eight MX derivatives were assayed. Several were studied together in a t least one assay. In addition to MX, the seven included 3-chloro-4-(dichloromethyl)-2(5H)-furanone(RMX, 2), 3-chloro4-(chloromethyl)-5-hydroxy-2(5H)-furanone(3), 3-chloro-4-(chloromethyl)-2(5H)-furanone(4), 4-(chloromethyl)-5-hydroxy-2(5H)-furanone( 5 ) , 4-chloromethyl-2(5H)-furanone (6), and 4(dichloromethyl)-2(5H)-furanone(8). Compounds 1-6 were mutagenic. Compound 8 gave erratic results. 4-(Acetoxymethyl)-2(5H)-furanone(11) was nonmutagenic. The largest drop in mutagenicity amounted to a factor of about lo2 for the replacement of the hydroxyl group or a C-3 chlorine atom from 3. Other replacements of the hydroxyl group or a C-3 or C-6 chlorine atom amounted to mutagenicity diminished by a factor of only 10. On the basis of the rates of UV spectral changes under assay conditions, chemical half-life values (? ) for 1-6 and 8 were estimated as indicators of compound stability. However, mutagenicity ifferences were shown to result principally from the intrinsic mutagenicities of the six compounds 1-6 rather than from differences in stability. Plots of mutagenicity against the I3C chemical shifts for C-2, C-3, and C-4(6cS2,6c.3, bC-!) in compounds 1-6 resulted in each case in a linear dependency. Plots of gave mutagenicity against 6c.z and 6c.4had negative slopes. The plot of mutagenicity against ijCs3 a positive slope. The correlations were attributed to the dependence of mutagenicity on electron density a t C-2,C-3,and C-4.
Y
Introduction This paper concerns the relative mutagenicity of compounds 1-11, all 2(5H)-furanones. It also concerns the development of a correlation between the mutagenicities of these compounds and their 13C NMR chemical shifts.
yx Z
0
5:W = X = H ,Y = CI;Z =OH 6:W = X = Z = H , Y = CI 7:W = H ,X = Y = c1,Z =OH
1:W = X = Y= C1;Z = OH
8: W = H X = Y = C1;2 = H
2:W=X=Y=Cl;Z=H
9:W = X = Y = H 2 = OH
3:W = Y =C1;X = H ,Z = OH
10:W = X = Y = Z = H
4:W = Y = C1;X = Z = H
11: W = X = Z = H ,Y = OAc
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, hereafter referred to as MX (l),is a direct acting mutagen that has been identified in softwood kraft chlorination effluent (1) and the chlorination of humic waters (2, 3). MX has been found in several samples of chlorine-disinfected drinking waters as well (4-8). The contribution of MX to the total mutagenicity of drinking water has been found to range from 3 to 33% ( 5 ) .
Scheme I
-
Clf
1
12
i c o o , CI 13
The mutagenicity of MX has been reported in the range of 103-104rev/nmol' in the assay with Salmonella typhimurium tester strain TA1W2lacking metabolic activation by rat liver homogenate fraction S9, (7, 9, 10). The importance of MX as a TAlOO mutagen is indicated by this high level of activity. Another indication of MX's genotoxicity is the observation that MX in the absence of S9 and at a dose as low as 4 pg/mL induced chromosomal aberrations in Chinese hamster ovary cells after 6-8 h of exposure (11). Additionally, treatment of bacterial and mammalian cell lines with MX has been reported to produce DNA-MX adducts (12), although the nature of the binding of the two components comprising the adducts is unknown. Recent papers dealing with the origin and genotoxicity of chlorinated 2(5H)-furanones and their open-ring forms Abbreviations of units rev/Kmol= revertants/micromole;rev/nmol
Department of Chemistry. Department of Paper Science and Engineering.
= revertants/nanomole.
Salmonella typhimurium tester strain TAlOO is referred to hereafter as TA100.
oa93-22a~/9~/270~-0035~02.50/0 0 1991 American Chemical Society
36 Chem. Res. Toxicol., Vol. 4, No.1, 1991 also attest to a n interest in relating the chemical structure of these compounds t o their mutagenicity. Tikkanen and Kronberg (13) have reported that (E)-2-chloro-3-(dichloromethyl)-4-oxo-2-butenoic acid (EMX, 13), t h e diastereomer of t h e open-ring tautomer of MX (121, could be no more t h a n 5 % as potent and probably was inactive (Scheme I). Another study (lo), wherein several 2(5H)-furanones were assayed under the standard conditions of t h e TAlOO Ames assay (14), showed that substitution of the 5-methoxy for the 5-hydroxy group of MX had virtually no effect on mutagenic potency. In the same study, however, removal of the two chlorine atoms from C-6, giving 14, diminished mutagenicity by a factor of lo3,
14: X =Cl;Y
C H 3 ; Z =OH
15: X = Y = C1;Z = O H
whereas removal of all chlorine atoms dropped t h e mutagenicity to roughly 10 rev/pmol, a level which can be questioned as mutagenic according to t h e criteria of Prival a n d Dunkel (15) for mutagenicity. I n more recent work, LaLonde and co-workers (16) demonstrated that t h e removal of the 5-hydroxyl group from mucochloric acid (15) virtually inactivated the latter. Similarly, in a study t o ascertain the relation of open- and closed-ring forms of MX on t h e mutagenicity of t h e latter, the replacement of t h e 5-hydroxyl group by a hydrogen atom resulted in roughly a 10-fold reduction of TAlOO m ~ t a g e n i c i t y . ~ One of our continuing interests in t h e 2(5H)-furanone family of mutagens has been to develop a structure-activity relationship that would point t o t h e minimum substitution patterns required for a high level of mutagenicity, defined here as being of t h e order of lo3 rev/nmol or greater, and would have predictive utility in assessing t h e Ames mutagenicity for members of t h e 2(5H)-furanone family, whose activity had not yet been determined. We show here how t h e stepwise removal of chlorine atoms and a hydroxyl group from t h e 4-methyl-2(5H)-furanone framework changes TAlOO mutgenicity. We also show that the mutagenicity correlates with 13C chemical shift d a t a for t h e three carbons ((2-2, C-3 a n d C-4) of t h e cu,p-unsaturated carbonyl system. Finally, we rationalize this correlation in terms of electron density at these three carbon centers.
Experlmental Procedures Chemicals. Samples of compounds (1-3 and 5-8 and 11) were obtained as previously described (17). Compound 4 was prepared from 6 through a simplified procedure analogous to the conversion of 8 to 1 (I 7). All samples were analyzed for purity by 'H NMR and capillary gas chromatography (GC), and TLC when useful. Samples were purified by flash chromatography (18)until the 'H NMR and GC showed that the sample was homogeneously one substance with a purity exceeding 99%. Mutagenesis Assay. The histidine-requiring (his-) S. typhimurium tester strain TAlOO supplied by Dr. Bruce Ames (University of California, Berkeley) was used in the standard plate incorporation mutagenesis assays performed according to Maron and Ames ( 1 4 ) for 72-h periods. The mutagenic activities of all compounds, added in freshly prepared DMSO solutions to the top agar, were determined without activation by rat liver homogenate fraction S9. Three plates per dose level were prepared, R. T. LaLonde, G. P. Cook, H. Perakyla, C. W. Dence, and J. G. Babish, unpublished studies.
LaLonde et al. and mutagenic responses were determined simultaneously for as few as three and as many as seven compounds in five assays, numbered 2, 6,7,8, and 9. The mutagenicity of one compound, 8, was determined singly in four assays, numbered 1, 3, 4, and 5, but also in assays 6 and 8. Additionally, prior assays had been carried out with the compounds to establish toxic dose levels in relation to the lower nontoxic dose/response levels. Values for mutagenicity as rev/pg or rev/ng were obtained as the positive linear regression slopes from the linear portion of the dose/response plots. The spontaneous TAlOO mutants, observed from the DMSO controls, were taken as the zero-dose points. Each assay included positive DMSO, crystal violet, ampicillin, and sodium azide controls. Bacterial controls were conducted in quintuplicate. 'H and 13C NMR and UV Spectra and Gas Chromatography. 'H NMR (60 MHz) were determined in CDC13 solution in 5-mm tubes on a Varian EM360 spectrometer. 13C NMR chemical shift values (6, TMS = 6 0.00) were taken from LaLonde and co-workers (17)and given linear least-squarestreatment with the mutagenicity levels. The UV spectra of 1-6,8, and 11 were recorded for the stability studies on Kontron UVIKON860 and Varian DMSlOO spectrometers. Solutions were prepared by dissolving a compound in DMSO and then diluting with Vogel-Bonner buffer solutions prepared according to the method of Maron and Ames (14). The final solution, examined by UV, contained the compound 15-49 mg/L and DMSO at 0.3-1.3% in a 1.00-cm quartz cuvette. Gas chromatography (GC) was performed on a Varian 3300 gas chromatograph equipped with a 30 m X 0.25 mm SPB-5 capillary column at 150 "C. The injector and FID detector were operated at 150 and 300 "C, respectively. The nitrogen flow was 1.1 mL/min. Stability Studies. Two flasks, the one containing the sample in DMSO-Vogel-Bonner buffer solution and the other, the control, containing only DMSO-Vogel-Bonner buffer solution, were kept at 37.5 f 0.1 "C. Aliquots were withdrawn simultaneously from both flasks at regular short intervals and placed in the spectrometer for UV absorbance determinations. Absorbance values were plotted against time to obtain the curves from which linear regression of two to five_successive points produced the derivative, (dA/dt)i, at the point Ai, determined to be the mean for the same two to five points. The slope obtained from the straight-line plot of (dA/dt)iagainst Ai gave the estimated values for the assumed first-order rate constants, k , from which the half-life values were calculated. The estimated error in the k was calculated as the s u m of the absolute values of the maximum positive and negative deviations from the regression straight line divided by the difference of the first and last mean absorbance values. Estimated were obtained from Ctli2= 0.693/k. chemical half life values (Ctli2)
Results Dose-response plots for t h e six compounds 1-6 were obtained in five different assays. I n four of these assays, two t o five of t h e six compounds were assayed simultaneously. At least one assay, assay 8, involved t h e simultaneous assay of all six of these compounds. T h e mutagenic response of no compound determined in t h e other four assays varied by more t h a n a factor of 3 from its response in assay 8. Because the mutagenic doses spanned a range of lo4, i t was necessary t o scale dose values from assay 8 by factors in order t o display responses of t h e six compounds simultaneously. Thus, Figure 1 is offerred primarily for t h e ready visual assessment of response linearity a n d revertant standard deviations, as expressed by t h e error bars. Linear regression treatment of the data of similar graphs, as exemplified by Figure 1, afforded t h e slopes in units of rev/ng or rev/pg for each of t h e several response plots. Multiplication of these slopes by t h e molecular weight in units of ng/nmol or pg/pmol gave the molar mutagenicities in units of rev/nmol or rev/pmol,_respectively. Finally, t h e mean molar mutagenicities (W-were calculated for each of six compounds. Values of M a r e given in Table I. An unabridged version of this table includes statistical
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 37
Mutagenicity of Chlorinated 2(5H)-Furanones 3600
T
3400
Table I. S.typhimurium TAlOO Mutagenicities and a Summary of Statistical Results from the Linear Regression Analysis of Several Assays of MX and Its Analogues Derived from 4-Methyl-2(5H)-furanone examined mean mean molar compd in assay nos. slopeo mutagenicity (I@) SDb 1 2, 6-8 17.6‘ 3840d 143P 0.88OC 177d 77.9e 2 2, 6, 8 3 7-9 3.16‘ 579d 86.5e 4 2, 7-9 29.d 49208 999h 5 7-9 26.lf 38708 731h 6 2,7-9 2.31f 30W 68h 8 1-6,8 9‘ 0.259’ 3wJ
L
3200
3000 2800 0 4-
9
$ 2
p E
2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200
o
1 J
! 0
l
~
l
200
400
600
~ 800
, 1000
L
~
1200
Dose in ng x f a c t o r
Figure 1. Dose-response plots for assay 8, for compounds 1 (m), 2 (O), 3 ( O ) ,4 (O),5 (A),and 6 (A). Standard deviations (SD) are represented by the vertical error bars, which are concealed when SD is less than the height of a symbol. The horizontal-axis units are in ng, which have been multiplied by a factor of 1 for for compounds 4 and 5; and for comcompounds 1-3; pound 6. Doses of compounds 4-6 were a t the microgram level but were converted to units of ng for uniformity of display and ease of comparison in this figure.
information relevant to each doseresponse determination and is available as supplementary material. No mutagenicity data is given in Table I for compound 8 since its assays gave erratic results. Consequently, a representative AT could not be calculated for 8. Also, no entries in Table I will be found for compounds 7 and 11. The former compound was judged from preliminary stability studies to be too unstable for assay. The latter compound was assayed but showed no mutagenic response up to 1.0 mg/plate dose levels and, therefore, was judged nonmutagenic by the criteria of Prival and Dunkel (15). Table I was extended to include data for compound 9, for comparison. This compound had been studied earlier in this laboratory (10). Finally, the mean molar mutagenicities are compared in Figure 2 without regard for compound stability differences (vide infra). Figure 2 illustrates, in the reciprocal factors associated with each small arrow, the diminishing mutagenicity resulting from the stepwise removal of chlorine atoms or the hydroxyl group starting from MX.
The mean value of the linear regression slopes from the assays indicated in column 2. Standard deviation. ‘Revertants/ng. In revertants/nmol and calculated by multiplying the linear regression slope by the molecular weight of the compound in ng/nmol. l e In revertants/nmol. ~ l ~ fRevertants/pg. l ~ #In ~revertants/pmol and calculated by multiplying the linear regression slope by the molecular weight of the compound in pg/nmol. hIn revertants/pmol. ’Results taken from ref 10. ”onmutagenic by the criteria of PreVal and Dunkel (15).
Table 11. Chemical Half-Life Values ( “ t l l zfor ) Compounds 1-6,8, and 11 Estimated under Conditions of Mutagenesis Assay by the Method Indicated under Experimental Procedures compd h ?& error X,O nm Ab data pointsC 1 11 38 269 31 2 2.6 22 235 7 3 44 31 285 +6 4 2.9 31 229 15 5 3.3 21 251 6 6 12 30 237 + 31 8 3.0 31 241 + 20
+
11
>50
The wavelength followed. Increasing (+) or decreasing (-) absorbance at the indicated wavelength was followed. ‘The number of data points.
Estimated values of the chemical half-life (‘tllz) for the compounds 1-6, 8, and 11 are given in Table 11. The Ct,lz values reflect the relative compound stabilities under mutagenesis assay conditions. The results established that the inactivity of compound 11 was attributable to its intrinsic nonmutagenicity rather than to its relative lack of stability. Relative to MX, the stability under assay conditions of the other six mutagenic compounds ranged from roughly one-forth as much (2) to four times greater (3). Thus, the stability spans a factor of roughly sixteen from least stable to most stable.
vo Lx0- lho-
1c1
CI
1/6.6_3
11150
1/129*
0
HO
“x0 - 4c1xo - 6c1h 1 I118
1 111.5
w
2
1136.0
0
,
......
1116.1
0
0
0
0
0
Figure 2. A comparison of the structures and mutagenicities of MX (1) and its 4-methyl-2(5H)-furanone (10) analogues by the stepwise removal of chlorine atoms (along the horizontal axis) and the hydroxyl group (along the negative vertical axis). Resiprocal number values as_sociatedwith the smaller arrows represent the factors diminishing the corresponding mean mutagenicities (M) given in Table I. The M values in units of rev/pmol were converted to units of rev/nmol for the purpose of calculating the factor shown. The asterisk (*) refers to a factor furnished in part by data from Ishiguro and co-workers (4). Dotted-line arrows represent comparisons of structure only.
LaLonde et al.
38 Chem. Res. Toxicol., Vol. 4, No. 1, 1991 4.0 4 3.5
3'0 2.5
1.
I
I
I
I
I
1
I I
I
i\
chemical shifts of a parLicular carbon (6cj), which included each of the five carbon atoms 0' = 2-6). Acceptable, least-sqaures, straight-line plots, shown in Figure 3, were obtained only for carbons 2-4, the carbons of the a,@-unsaturated carbonyl system. The regression equations are log M = -0.5256c.2
+ 89.7
log A = 0.5086c.3 - 60.2 log M = -0.2116c.4
-0.5 O.O -1.0
166
4.0 I
-
I
118
1
167
168
I
I
171
170
169 I
I
172 I
173 I
6
-1.0
3.5
I
I
I
I
I
I
119
120
121
122
123
124
1 C
125
i-
t
c
150
155
160
165
Figure 3. Plots of chemical shifts, bc.j (horizontal axis), against the log mean_mutagenicity, log M (vertical axis), including bca against log M (a), dc.3 against log M (b), and b ~ against . ~ log M (c), for compounds 1-6.
Following the type of quantitative structure-activity relationships (QSAR) pioneered by_Hansch; the log values of the mean mutagenicities (log A4), in rev/nmol, of each of the six compounds 1-6 were plotted against the 13C 'See
(2)
(3)
The squared correlation Coefficients were 0.835 (P= 0.011), 0.766 ( P = 0.022), and 0.678 ( P = 0.044), respectively, for the straight lines represented by eqs 1, 2 and 3. No significant change in slopes, intercepts, or squared correlation coefficients were observed when M values were replaced by the mutagenicity values from which M values were calculated.
iI 165
-0.5
+ 34.5
(1)
ref 19 for a recent example of this type of QSAR applied to
TAlOO mutagenicity.
Discussion and Conclusion Without regard for the relative assay stabilities of compounds 1-6, Figure 2 would denote that the stepwise replacement of hydrogen for a chlorine atom or a hydroxyl group effects a diminished mutagenicity at each step. However, as the data of Table I1 demonstrate, estimated Ct,/zvalues of compounds 1-6 differ. Possibly, the greater mutagenicity of a compound may be associated with a value, implying that the greater mutagenicity higher Yll2 stems from a higher concentration of the mutagen in a critical period (tcp) for incipient reversion. For example, such a case might be made for compounds 1-4. Both mutagenicity and Ctl/zare larger for the two compounds 1 and 3, which possess both a C-3 chlorine atom and a C-5 hydroxyl group, than they are for compounds 2 and 4, which lack a C-5 hydroxyl group. In contradistinction, compounds 5 and 6 lack a C-3 chlorine atom. In this case, higher mutagenicity is not associated with the higher Ctl12 value. A rationale for the coincidence of high mutagenicity and Ctllzvalues in the case of compounds 1-4 and the lack of such coincidence in the case of 5 and 6 would be that the negative inductive effect of the C-3 chlorine atom stabilizes the carboxylate ion of the open-ring form of mutagens 1 and 3, as exemplified by the carboxylate ion corresponding to structure 12 (Scheme I). The carboxylate anion of the open-ring form would be less reactive with inactivating nucleophiles than the ring form of the mutagen. Although the stability-based rationale of the preceding paragraph could account qualitatively for the factors separating the mutagenicities of compounds 1-4, the following discussion leads us to the conclusion that stability differences alone are of little significance to the mutagenicity reported here. This discussion concerns relative rates of mutagenic decay. Arguments have been educed3 that the number of mutagenic events can be related to the area under the curve for a first-order decay of mutagen concentration. Equation 4 reproduces the relationship
between the area S and the initial mutagen concentration (c,,), the chemical half-life (YlI2), and the critical period (tCJ for incipient mutagenesis. Moreover, the ratio of mutagenic events initiated by two mutagens was considered to be related to the ratio of the two areas (Sl/Sz) under the curves. From the latter relationship, it was shown that the limiting upper ratio of relative mutagenesis stemming from different stabilities could be expressed simply as the ratio of the two Ctllzvalues, when t,, was
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 39
Mutagenicity of Chlorinated 2(5H)-Furanones Scheme I1
carbonyl carbon in these compounds (25). Therefore, the enhanced mutagenicity of MX, relative to other members of the series, is associated with higher electron density at C-2 and C-4 and lower electron density at C-3. That the correlation represented by eq 1 includes only a single parameter indicates that other molecular properties, such as hydrophobic character, are relatively unimportant for the mutagenicity of these compounds or, possibly, that small differences in hydrophobicity between compounds precludes it being an important factor. This study demonstrates how S. typhimurium TAlOO mutagenicity responds to location and degree of chlorine and hydroxyl group substitution in a group of 2(5H)furanones. The study considers varying stability as a possible explanation for differences in mutagenicity but rejects this as an explanation on the argument that the relative differences in mutagen decay have to be many times less than the observed differences in mutagenicity. The ensuing development of a linear relationship between mutagenicity and 13C chemical shifts is attributed to the response of mutagenicity to the electron density in the domain of the a$-unsaturated carbonyl system.
6h-h-h 0 16
0
17 0
0’
0 18
0’
taken as infinity. When this limiting case is applied to compounds 3 and 4, 3 would be no more than 15 times more mutagenic than 4. If t, for the bioassays is chosen as 5 h, then compound 3 woufd be no more than 1.7 times as mutagenic as 4. According to experimental results, however, 3 is slightly more than 100 times more mutagenic than 4. The difference between Ct,lzvalues of the other linked mutagens of Figure 2 is less than that between 3 and 4, and accordingly, the effect on mutagenesis stemming from mutagen stability differences will be smaller also. We conclude, therefore, that the stepwise reduction of mutagenicity that is summarized in Figure 2 represents changes in the intrinsic mutagenicities resulting from the substitution of hydrogen for chlorine atoms or hydroxyl groups. This being the case, the replacement of the hydroxyl group or a C-3 chlorine atom in 3 by hydrogen diminishes mutagenicity by factors slightly exceeding lo2. The succeeding replacement of a C-6 chlorine atom by hydrogen in 5 giving 9 diminishes mutagenicity again by a factor of lo2 and reduces activity to near nonmutagenicity. However, the comparison of mutagenicity for 5 and 9 might be considered tentative since the two compounds were not assayed together. All other hydrogen for chlorine atom or hydroxyl group replacements diminish mutagenicity by factors of the order of 10. Therefore, 3 represents an elevated stratum in mutagenicity values from which additional chlorine substitution, resulting in MX (l),leads to only a modest increase. The data set, from which eqs 1-3 were obtained, is still quite small. Nevertheless, the correlation coefficient for eq 1 is large enough to lend confidence to the use of the relationship for assessing the mutagenesis level of other small, halogenated 2(5H)-furanones. We are unable to explain the erratic mutagenicity results obtained for 8, which we believed to be homogeneous on the basis of ‘H NMR and GC. However, the application of eq 1 and the known 6c.z value for 8 (17) indicates this compound could be expected to exhibit mutagenicity of the order of lo3 rev/pmol. A similar treatment of the unstable 7 advises one that this compound’s mutagenicity would be of the order of lo4 rev/pmol. We attribute the correlations between log and 6c.j to the response of mutagenicity to relative electron densities at C-2, -3, and -4 of the series of 2(5H)-furanones. Several theoretical and empirical treatments have indicated a relation between chemical shift values and electron density (20-23). Larger and smaller chemical shift values have been linked respectively to lower and higher electron density. Significantly, plots of log M against 6c-z and 6c.4 values gave negative slopes while the plot of log M against the Bc.3 values gave a positive slope. We believe the electron density at the three carbon centers can be explained through the interplay of three factors: electronic polarization of the a,@-unsaturatedcarbonyl system, as depicted in canonical structures 16-18 (Scheme 11); electronic suppression of this system’s polarization by the presence of the electron-withdrawing C-3 chlorine atom; and augmented suppression of this system’s polarization by increasing substitution at C-5 and C-6 by hydroxyl group and chlorine atoms. The suppression of ketone polarization in simple a-haloketones and aldehydes (24, 25) has been given to explain the relative shielding of the
Acknowledgment. Research supported by the US. Geological Survey, Department of the Interior, under USGS Award 14-08-0001-G1498. The views and conclusions contained in this paper are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the US. Government. We thank the Sterling-Winthrop Research Institute for the support of the several undergraduates who assisted this research through the preparation of intermediates employed in the synthesis of the assayed compounds. Registry No. 1, 77439-76-0;2, 122551-89-7; 3, 125974-08-5; 4,125974-01-8; 5,125974-06-3; 6,125973-99-1;8,125974-00-7; 11, 81189-55-1.
Supplementary Material Available: An unabridged version of Table I, giving statistical information relative to each doseresponse determination (4 pages). Ordering information is given on any current masthead page.
References (1) Holmbom, B. R., Voss, R. H., Mortimer, R. D., and Wong, A. (1984)Fractionation, isolation and chlorination of Ames mutgenic compounds in kraft chlorination effluents. Enuiron. Sci. Technol. 18, 333-337. (2) Kronberg, L.Holmbom, B., and Tikkanen, L. (1985)Fractionation of mutagenic compounds found during the chlorination of humic water. Sci. Total Enuiron. 48, 343-347. (3) Meier, J. R., Ringhand, H. P., Coleman, W. E., Munch, J. W., Streicher, R. P., Kaylor, W. H., and Schenck, K. M. (1985)Identification of mutagenic compounds formed during chlorination of humic acid. Mutat. Res. 157, 111-122. (4) Kronberg, L., Holmbom, B., and Tikkanen, L. (1985)Mutagenic activity in drinking water and humic water after chlorine treatment. Vatten 41, 106-109. (5) Hemming, J., Holmbom, B., Reunanen, M., and Kronberg, L. (1986)Determination of the strong mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in chlorinated drinking and humic waters. Chemosphere 5,549-556. (6) Kronberg, L., Holmbom, B., Reunanen, M., and Tikkanen, L. (1988)Identification and quantification of the Ames mutagenic compound 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-fur~one and of it,s geometric isomer (E)-2-chloro-3-(dichloromethyl)-4oxobutenoic acid in chlorine-treated humic water and drinking water extracts. Enuiron. Sci. Technol. 22, 1097-1103. (7) Meier, J. R., Knohl, R. B., Coleman, W. E., Ringhand, H. P., Munch, J. W., Kaylor, W. H., Streicher, R. P., and Kopfler, F. C. (1987) Studies on the potent mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxyl-2(5H)-furanone; aqueous stability, XAD recovery and analytical determination in drinking water and in
40 Chem. Res. Toxicol., Vol. 4, No. 1, 1991 chlorinated humic acid solutions. Mutat. Res. 189, 363-373. (8) Meier, J. R. (1988)Genotoxic activity of organic chemicals in drinking water. Mutat. Res. 196, 211-245. (9) Holmbom, R., Voss, R. H., Mortimer, R. D., and Wong, A. (1984) Fractionation isolation and chlorination of Ames mutagenic compounds in kraft chlorination effluents. Enuiron. Sci. Technol. 18, 333-337. (10) Ishiguro, Y.,LaLonde, R. T., Dence, C. W., and Santodonato, J. (1987)Mutagenicity of chlorine-substituted furanones and their inactivation by reaction with nucleophiles. Enuiron. Toxicol. Chem. 6,935-946. (11) Meier, J., Blazak, W., and Knohl, P. (1987)Mutagenic and clastogenic properties of 3-chloro-4-(dichloromethyl)-5-hydroxy2(5H)-furanone: A potent bacterial mutagen in drinking water. Enuiron. Mutagen. 10, 411-424. (12) Meier, J. R. (1989)DNA adduct formation in bacterial and mammalian cells by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Enuiron. Mol. Mutagen. 14 (Suppl. 15), 128. (13) Tikkanen, L., and Kronberg, L. (1990)Genotoxic effects of various chlorinated butenoic acids identified in chlorinated drinking water. Mutat. Res. 240, 109-116. (14) Maron, D. M., and Ames, B. N. (1983).Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173-215. (15) Prival, M. J., and Dunkel, V. C. (1989)Reevaluation of the mutagenicity and carcinogenicity of chemicals previously identified as "false positives" in the Salmonella typhimurium mutagenicity assay. Enuiron. Mol. Mutagen 13, 1-24. (16) LaLonde, R.T., Perakyla, H., Cook, G. P., and Dence, C. W.
LaLonde et al. (1990)The contribution of the 5-hydroxyl group to the mutagenicity of mucochloric acid. Enuiron. Tonicol. Chem. 9, 687-691. (17)LaLonde, R. T., Perakyla, H., and Hayes, M. (1990)Potentially mutagenic, chlorine-substituted 2(5H)-furanones: Studies of their synthesis and NMR properties. J. Org. Chem. 55, 2847-2855. (18) Still, W. C., Kahn, N., and Mitra, A. (1978)Chromatographic technique for preparative separations with moderate resolution. J. Org. Chem. 43, 2923-2925. (19) Lopez de Compadre, R. L., Debnath, A. K., Shusterman, A. J., and Hansch, C. (1990)LUMO energies and hydrophobicity as determinants of mutagenicity by nitroaromatic compounds in Salmonella typhimurium. Enuiron. Mol. Mutagen. 15, 44-55. (20) Karplus, M., and Pople, J. A. (1963)Theory of carbon NMR chemical shifts in conjugated molecules. J. Phys. Chem. 38, 2803-2807. (21) Ditchfield, R., and Ellis, P. D. (1974)Theory of I3C chemical shifts. In Topics i n Carbon-13 N M R Spectroscopy (Levy, G., Ed.) Vol. 1, pp 1-51, Wiley, New York. (22) Loots, M. J., Weingarten, L. R., and Levin, R. H. (1976)Application of 13Cnuclear magnetic resonance spectroscopy to the analysis of charge distribution patterns in unsaturated carbonylcontaining compounds. J. Am. Chem. SOC.98, 4571-4577. (23)Spiesecki, H., and Schneider, W. G. (1961)The determination of *-electron densities in azulene from C13 and H' nuclear resonance shifts. Tetrahedron Lett., 468-472. (24) Breitmaier, E.,and Voelter, W. (1987)Carbon-13 N M R Spectroscopy, pp 216-222,VCH Publishers, New York. (25) Stothers, J. B. (1972) Carbon-13 N M R Spectroscopy, pp 282-303,Academic Press, New York.
