Chem. Res. Toxicol. 1991,4, 535-540
535
Microcystins from Anabaena flos-aquae NRC 525-17 Ken-ichi Harada,* Kiyoshi Ogawa, Yukio Kimura, Hideaki Murata, and Makoto Suzuki F a c u l t y of P h a r m a c y , Meijo University, T e m p a k u , Nagoya 468, Japan
Patti M. Thorn, William R. Evans, and Wayne W. Carmichael Department of Biological Sciences, Wright S t a t e University, Dayton, Ohio 45435 Received F e b r u a r y 25, 1991
Anabaena flos-aquae NRC 525-17 produces a very potent neurotoxin, anatoxin-a(s). During isolation of the neurotoxin, we found that the strain contains four other toxic compounds which show strong hepatotoxicity. The four toxins, toxins 1, 1', 2, and 3, were successfully purified. Toxin 2, one of major toxins, was identified as 3-desmethylmicrocystin LR (1) by comparison of spectral data of the known compound. Since the three other toxins contain an unknown amino acid, GC/MS was applied and it revealed the presence of homotyrosine in toxins 1 (2) and 1' (3). Only a partial structure was obtained for toxin 3 due to the small amount present in the cells.
I ntroductlon
Experimental Procedures Materials. A. flos-aquae NRC 525-17 was cultivated according
The occurrence of toxic freshwater blooms of cyanoto the method described in Mahmood and Carmichael's method bacteria has been reported in many countries. Toxins from (22). Cells were lyophilized prior to toxin extraction. freshwater cyanobacteria are classified into two groups, Separation of Toxins 1, l', 2, and 3. Dried cells (20 g) were neurotoxins and hepatotoxins (1-3). The coccoid genus extracted with 0.05 M AcOH-EtOH (400 mL) three times for 3 Microcystis aeruginosa is the most common toxin-proh while stirring. The combined extracts were centrifuged at 3500 ducing cyanobacterium found worldwide, and it produces rpm for 15 min, and the supernatant was evaporated to dryness. potent cyclic peptide hepatotoxins termed microcystins The residue was dissolved in water (300 mL), and the solution was centrifuged at loo00 rpm for 1 h. The supernatant was (1-5). Microcystins are also found in Microcystis viridis applied to a preconditioned ODS silica gel column (22 X 300 mm, (6-8), Anabaena flos-aquae (9),Oscillatoria agardhii ( l o ) , Chromatorex ODS, Fuji-Davison Chemical Ltd., Tokyo, Japan). and Nostoc sp. ( 1 1 ) . Nodularia spumigena grows mainly The column was successively rinsed with HzO (250 mL) and 20% in brackish water and produces another hepatotoxin, noMeOH-H20 (250 mL) and then eluted with MeOH (250 mL) to dularin. Microcystins are characteristic cyclic heptagive a fraction (170 mg)containing the hepatotoxins. The residue peptides composed of y-linked D-glUtamiC acid (Glu), Dof the 0.05 M AcOH-EtOH extraction was reextracted with 5% alanine (Ala), @-linkedD-erythro-@-methylasparticacid AcOH to give 192 mg of the crude toxin-containingfraction. The (P-Me-Asp), N-methyldehydroalanine (Mdha),' a novel combined toxic fraction was chromatographed on silica gel (20 @-amino acid, 3-amino-9-methoxy-lO-phenyl-2,3,8-tri- X 420 mm, 230-400 mesh, Nacalai Tesque, Kyoto, Japan) using CHC13/MeOH/Hz0 (65255, lower phase) to yield a 6-mg fraction methyldeca-4,6-dienoic acid (Adda), and two variable Lcontaining toxins 1and 1' and a 61-mg fraction containing toxins amino acids (12, 13). Over 10 microcystins have been 2 and 3. The first fraction was separated by preparative HPLC isolated so far (1-3,5,14,15). Nodularin is a pentapeptide using ODS silica gel to give 1.3 mg of toxin 1and 0.6 mg of toxin similar to microcystins (16, 17). Recently, it has been 1'. The second was separated by ODS silica gel chromatography found that microcystins and nodularin inhibit protein [ l l X 940 mm, Chromatorex; flow rate, 0.5 mL/min; detection, phosphatase activity, especially 1 and 2A, in a manner UV (238 nm)] using MeOH/0.05 M sodium sulfate (1:l)as the similar to that of okadaic acid (18-20). mobile phase to give 4.6 mg of toxin 2 and 0.4 mg of toxin 3. Each Three neurotoxins have been isolated to date. Aphanisolated component was finally purified by using TOYOPEARL toxins (saxitoxins) are produced by A p h a n i z o m e n o n HW-4OF gel chromatography [ l l X 970 mm, Tosoh, Tokyo, Japan; flow rate, 0.4 mL/min; detection, UV (238 nm)] with MeOH as flos-aquae (21),and anatoxin-a and -a(s) are produced by the mobile phase to yield toxins 1(1.0 mg), 1' (0.4 mg), 2 (2.8 mg), A n a b a e n a flos-aquae. Anatoxin-a is an alkaloid-like and 3 (0.2 mg). compound (22)that has been synthesized ( 1 ) . The third HPLC. The following two HPLC systems were used for neurotoxin is anatoxin-a(s) produced by A. flos-aquae analysis of (I) microcystins and (11) absolute configuration of NRC 525-17, which has been found to be a potent anticonstituent amino acids. (I)A constant-flow pump (880-PU Jasco, cholinesterase (23, 24). In 1989 the structure has been Tokyo, Japan) was used. Separation was carried out under recompletely determined to be a unique phosphate ester of versed-phase isocratic conditions with a Nucleosil5C18 column a cyclic N-hydroxyguanidine (25). (4.6 X 150 mm, Chemco Scientific Co., Osaka, Japan). Two mobile We have also been studying toxins from this strain, phases, MeOH/0.05 M phosphate buffer (pH 3) (58:42) and particularly, the establishment of an analysis and purifiMeOH/0.05 M sodium sulfate (pH 7) (55:45) were used for analysis and preparative separation, respectively. The flow rate cation method and t h e structure determination of anatwas 1 mL/min, and the detection with UV (238 nm) was done oxin-ab). During separation of anatoxin-a(s), we found that this strain produces other toxins. In this paper we describe the isolation and structures of three hepatotoxins Abbreviations: Adda, 3-amino-9-methoxy-lO-phenyl-2,3,8-trimethyldeca-4,6-dienoicacid; Mdha, N-methyldehydroalanine;ODs, ocand partial structures of a fourth from A. flos-aquae NRC 525-17.
* Address correspondence to this author.
tadecylsilanized;FABMS, fast atom bombardment mass spectrometry; EIMS,electron ionization mass spectrometry; CIMS,chemical ionization mass spectrometry; FDAA, 2-[(5-fluoro-2,4-dinitrophenyl)amino]propanamide; N-TFA-n-Bu ester, N-(trifluoroacety1)-substitutedO-nbutyl ester; TFA, trifluoroacetic acid.
1991 American Chemical Society
Harada et al.
536 Chem. Res. Toxicol., Vol. 4,No.5, 1991 with a Jasco 875 UV detector. (11) The resulting amino acid derivatives were analyzed under the following gradient conditions: pump, CCPD (Tosoh, Tokyo Japan); system controller, PX-8010 and GE-8000 (Tosoh); degasser, DG-1300 (Uniflows, Tokyo, Japan); UV detector, 655A (Hitachi, Tokyo, Japan); column, Cosmosil5Cl8 (4.6 X 150 mm) (Nacalai Tesque, Kyoto, Japan). A linear gradient of (A) 90% triethylammonium phosphate (50 mM, pH 3)/10% CH3CN and (B) CH3CN with 0% B a t the start 40% B over 60 min (flow rate 1.5 mL/min) was used, and the derivatives were detected by UV absorption a t 340 nm. In the case of separation of D-Ala and erythro-P-Me-Asp, an isocratic condition, CH3CN/50 mM ammonium acetate (pH 3) (2:8), was used. Amino Acid Analysis. Purified toxins were hydrolyzed in 6 N HCl a t 106 "C for 24 h prior to amino acid analysis. The released amino acids were analyzed with a Waters (Milford, MA) Pic0 Tag HPLC system. The absolute configurations of the released amino acids were determined according to Marfey's method (26). To a 1-mL vial containing the hydrolysate in 200 pL of H 2 0 from each microcystin (200 pg) was added 100 pL of 1% of 2-[(5-fluoro-2,4-dinitrophenyl)amino]propanamide(FDAA) in acetone followed by 20 pL of 1N NaHCO3 The mixture was heated for 1 h a t 40 "C. After cooling to room temperature, 20 pL of 1N HCl was added and the resulting solution was subjected to HPLC analysis. GC/MS. Prior to analysis each purified toxin (100 pg) was hydrolyzd in 6 N HCl a t 110 "C for 15 h. The hydrolyzed mixture was evaporated to dryness, and the residue was reacted with 10% hydrogen chloride in n-BuOH (100 pL) a t 110 "C for 15 min. The reaction mixture was evaporated to dryness. The residue was heated in TFA (100 pL) a t 150 OC for 10 min, and the reaction mixture was evaporated to dryness. The residue was dissolved in 50 pL of CH2C12,and 1 pL of the solution was subjected to GC/MS analysis. Operating conditions were as follows: instrument, Shimadzu GC/MS QP 1000 (Kyoto, Japan); injector, Shimadzu CLH-702; injector temperature, 280 "C; column temperature, 100 280 "C (5 "C/min); carrier gas, He (40 mL/min); makeup gas, He (20 mL/min); separator temperature, 250 "C; ion source temperature, 250 "C; ionization energy, 70 eV for EIMS and 200 eV for CIMS; reagent gas for CIMS, isobutane; mass range, m/z 50-450 (EI) and m/z 100-550 (CI); interval time, 1.5 s; analytical time, 2-23 m h column, J&W Scientific (Folsom, CA) MEGABORE DB-5 (1.5 mm X 30 m). Spectral Analysis. FAB mass spectra were obtained with a double-focusing JEOL (Tokyo, Japan) JMS-HX 110 mass spectrometer. The fast beam was operated a t 6 kV with xenon gas, and the spectrometer was operated a t a 10-kV accelerating potential. Samples were dissolved in MeOH a t a concentration of 10 pg/pL, and samples (0.5-1.0 pL) were loaded. About 1pL of glycerol/l N HCl (1:l)matrix was added to the sample on a stainless steel target. 'H NMR spectra were recorded on a 270MHz JEOL JNM-GX 270 spectrometer using tetramethylsilane as an internal standard. Samples were dissolved in CD30D or CD30D containing a small amount of TFA. Specific rotations were measured with a Jasco DIP-181 polarimeter. Ultraviolet (UV) spectra were taken on a Shimadzu UV 2100 double-beam spectrophotometer. Determination of Toxicity. Toxicity of lyophilized cells, crude extracts, and purified compounds was examined by using the intraperitoneal (ip) mouse bioassay. Signs of poisoning were observed over a 24-h period and compared with those produced by microcystins (3, 9). This includes the blood-engorged hemorrhagic liver that occurs within 30-60 min after injection and is the key characteristic used to identify microcystin toxicosis. The 50% lethal dose (LDW)was estimated with five dose levels and six animals per dose level. After death, mice (18-23 g; male ICR Swiss) were autopsied and liver weights were determined as a percentage of body weight. This allows comparison of the liver weight with known cyclic peptide microcystins, all of which cause a characteristic increase to &12% compared with normal mouse liver weights of 4-6%. -+
-
Results and Discusslon Lyophilized cells of A. flos-aquae NRC 525-17 were extracted with 0.05 M AcOH-EtOH, and the extract was
I
0
"
'
'
1
5
'
'
5
'
I
10
'
.
'
I
"
"
1
15
2 0 min
Figure 1. High-performance liquid chromatogram of the MeOH fraction after cleanup with ODS silica gel. Four toxic peaks appear in the chromatogram. Column: Nucleosil5C18. Mobile phase: MeOH/0.05 M phosphate buffer (pH 3) (5842). Flow rate: 1 mL/min. Detection: 238 nm.
H ~ N ' 'N'
H 1 : R1 = CH(CH3)2, 2 : R I = CHZ-CEH4-p-OH, 3 : R I = CHZ-CEH4-p-OH, 4 : R1 = CH(CH3)2, 5 : R I = CEH4-p-OH,
R2 = H R2 = H
R2 = CH3 R2 = CH3 R 2 = CH3
Figure 2. Structures of toxins 2 (I), 1 (2), and 1' (3), microcystin LR (4), and microcystin YR (5).
evaporated to dryness. The residue was dissolved in water, and after filtration the supernatant was applied to ODS silica gel. The resulting aqueous fraction contained expectedly anatoxin-ab), whereas the MeOH fraction showed hepatotoxicity. Since the extraction was not complete for isolation of the hepatotoxins, the residue was reextracted with 5 % AcOH and the extract was adsorbed on ODS silica gel. The MeOH eluate was combined with the previous MeOH fraction. The toxic fraction was purified according to our method (27,28) for microcystins to give four toxic components, as shown in the high-performance liquid chromatogram (Figure 1). These are abbreviated toxins 1, l', 2, and 3, and toxins 1and 2 were major components. Toxin 1' has similar toxicity to those of microcystins LR (4) and YR (5) (LDW80-100 pg/kg, ip, mouse). The toxicity of toxins 1 and 2 is less toxic (LDW160-300 pg/kg). The physicochemical properties of these toxins are shown in Table I. Toxin 2 was obtained as an amorphous powder. FABMS showed that the toxin has a molecular weight of 980. Amino acid analysis gave Glu, Ala, Leu, Arg, Aap, and N-methylamine. These data suggested strongly that toxin 2 is a desmethylated microcystin LR. Direct comparison of this toxin with an authentic sample showed that toxin 2 is 3-desmethylmicrocystin LR (1) (5,29,30), in which 0-Me-Asp in 4 (31) is replaced by Asp (Figure 2). We have established a nondestructive method using a combination of two-dimensional NMR techniques for structural determination of microcystins (32). The method was successfully used to assign all protons and carbons in
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 537
Microcystins from Anabaena flos-aquae
'3
12
Mdha
Adds
Figure 3. 'H NMR spectrum (270 MHz) of toxin 1in CD30D obtained on a JEOL JNM-GX 270. The spectrum shows that the toxin possesses a para-substituted benzene ring in addition to Adda and Mdha.
y
2
(C)
HOoCH2-C-COOH CH3
Figure 4. Proposed structures for an unknown amino acid in toxin 1. Table I. Physicochemical Promrties of Toxins 1, l', 2, and 3 toxin 1 toxin 1' toxin 2 toxin 3 appearance
white powder [a]lbD (MeOH) -76.0' (c 0.25) FABMS, m / z 1045 (glycerol + (M+ HI+ 1 N HCl) GZH,nm 230,238 (log 4 (4.61,4.59)
white powder -92.0' (c 0.025) 1059 (M + H)+
white powder -78.6' (c 0.37) 981 (M + H)+
230,238 238 (4.79,4.76) (4.50)
white powder -32.0' (c 0.05) 1015 (M + H)+
238 (4.60)
microcystins (5,321. Although the method was applied to the unknown toxins in the present study, it was unsuccessful due to limited amounts of the toxins. Particularly, it was very difficult to obtain good-quality NMR spectra and to use other techniques. Figure 3 shows the 'H NMR spectrum for toxin 1,and the 'H NMR spectral data for olefinic protons are summarized together with those of toxin 1' and 5 (Table 11) (31). These assignments for the lower field region were consistent with those of microcystins that were completely assigned by the method mentioned above (5,32). These results indicate that toxins 1 and 1' also have Adda and Mdha as well as known microcystins (16). Additionally, four protons appear in the
- I
356
A
519
. . . . . . . . . . . . . . . . . . . . . . 5
10
15
20
miti
Figure 5. GC/MS analysis for hydrolysis products of toxin 1.
(a) Total ion chromatogram of the derivatized hydrolysis products. Arrow indicates the unknown amino acid. (b) Mass chromatogram of the derivatized hydrolysis products. The values of m/z 242, 342, 356, and 519 correspond to the derivatized Ala, Asp, Glu, and Arg, respectively. The mass chromatogram indicates that the molecular weight of the unknown amino acid is 195.
lower field region, which probably corresponds to a parasubstituted benzene ring. So toxins 1and 1' possess two benzene rings in the molecule. The molecular weight of toxin 1,1044, was confirmed by FABMS. The molecular weight is the same as that of 5, which contains Glu, Mdha, Ala, Tyr, B-Me-Asp, Arg, and Adda as constituent amino acids. However, toxin 1was found not to be identical with 5 by HPLC analysis, and the usual acid hydrolysis gave an unknown amino acid in addition to Asp, Glu, Arg, Ala, and N-methylamine. These results indicated that toxin 1 has a homo-type tyrosine, for example, (A), (B),or (C)
Harada et al.
538 Chem. Res. Toxicol., Vol. 4, No. 5, 1991
Table 11. ‘HNMR SDectral Data for Olefinic Protons of Toxins 1 and 1’ and Microcvstin YR chemical shift, ppm assignment toxin 1 toxin 1’ microcystin YR Adda 7 5.42 (1 H, d, J = 10 Hz) 5.42 (1 H, d, J = 10 Hz) 5.42 (1 H, d, J = 10 Hz) 5.45 (1 H, dd, J = 16, 10 Hz) Adda 4 5.45 (1 H, dd, J = 16, 10 Hz) 5.45 (1 H, dd, J = 16, 10 Hz) Mdha 3 5.48 (1 H, s) 5.47 (1 H, s) 5.49 (1 H, s) Mdha 3 5.95 (1 H, s) 5.93 (1 H, s) 5.89 (1 H, s) Adda 5 6.24 (1 H, d, J = 15.1 Hz) 6.24 (1 H, d, J = 15.1 Hz) 6.26 (1 H, d, J = 15.5 Hz) 6.65 (2 H, d, J = 8.1 Hz) 6.64 (2 H, d, J = 8.7 Hz) aromatic proton 6.66 (2 H, d, J = 8.4 Hz) 7.01 (2 H, d, J = 8.4 Hz) 7.00 (2 H, d, J = 8.4 Hz) 7.16-7.28 (7 H, m) 7.12-7.27 (5 H, m) 7.16-7.33 (5 H, m) I 157
m/z
Figure 6. E1 mass spectrum of the derivatized unknown amino
acid from toxin 1 obtained on a Shimadzu GC/MS QP 1O00. The ion at m / z 203 is derived from the cleavage at the benzylic position.
as shown in Figure 4, instead of tyrosine in 5. GC/MS is frequently used for identification of constituent amino acids of peptides and was applied for the structural determination of the unknown amino acid. The hydrolyzed amino acids of toxin 1 were derivatized to their N-(trifluoroacety1)-substituted 0-n-butyl esters (N-TFAn-Bu ester) (33),and then the CI and E1 mass spectra of the derivatized amino acids were measured. Each amino acid can be detected by the mlz value of protonated molecule, (M + H)+, in the mass chromatogram under CI conditions. There are five peaks of the constituent amino
acids in the total ion chromatogram of toxin 1 (Figure 5). The four peaks correspond to known amino acids, Ala, Asp, Glu, and Arg. The molecular weight of the remaining unidentified peak was confirmed to be 443 by the mass chromatogram, which corresponds correctly to that of the derivatized homo-type tyrosine, (A), (B), or (C).Candidate C is only commercially available. We prepared its NTFA-n-Bu ester and compared it with that of the natural sample, so that we could eliminate (C) for the unknown amino acid. In order to determine the correct structure as (A) or (B), we carefully investigated the E1 mass spectrum. The molecular ion is not found at mlz 443, but many important fragment ions are observed (Figure 6). The fragmentation patterns of the N-TFA-n-Bu ester of an amino acid under E1 conditions have been already established as shown in Figure 7 (34,35). This scheme was applied to our case. The ions at mlz 342, 227, 171,153, 69, and 57 are common to both structures (A) and (B). It is well-known that the benzylic position is easily cleaved to give a stable tropylium type ion (34). In the case of (A) the mlz 203 ion is formed, but the ion at mlz 217 is produced in the case of (B). In the E1 mass spectrum only the ion at mlz 203 was observed. This result strongly indicates that this homo-type tyrosine has the structure A. To our knowledge, it is the first case to isolate naturally
d z 342
d z 342
I
?
m/; 217
CH3-CH
’
I
I
6- S-t CF3 01
L
d z 69
d z 153
4
d z 227
nVrz 171
Figure 7. Fragmentation schemes for the N-(trifluoroacety1)-substituted0-n-butyl ester of the unknown amino acid under E1 condition. This result indicates that the correct structure is (A) for the unknown amino acid in toxins 1 and 1’.
Microcystins from Anabaena flos-aquae occurring homotyrosine (36). Very recently, Namikoshi et al. have found homoarginine in a microcystin isolated from the filamentous cyanobacteriumNostoc sp. strain 152 (37).
With use of Marfey’s method (26) the absolute configurations of the constituent amino acids, Glu and Asp, were determined to be D, and that of Arg was determined to be L. Since erythro-0-Me-Asp and Ala were coeluted under these conditions, both were separated with a modified mobile phase, CH3CN/0.05 M AcONH4 (adjusted to pH 3 with TFA) (2:8) and the absolute configurations were determined to be D. Because homotyrosine has not been commercially available, the epimerization was tried. Under prolonged acidic hydrolysis conditions, the epimerization of homotyrosine proceeded to give a pair of peaks (19:l). The peak newly found had longer retention time, indicating that the absolute configuration of homotyrosine is L. Thus, the structure of toxin 1can be deduced as 2 with reference to those of known microcystins. The physicochemical properties of two minor components, toxins 1’and 3, are also shown in Table I. The ‘H NMR spectra suggest that both toxins are also microcystin-related compounds. GC/MS analysis for acid hydrolysis products of toxin 1’ shows that the toxin has Ala, 0-Me-Asp, Glu, Arg, and homotyrosine as the constituent amino acid. In consideration of the structure of toxin 1, the molecular weight, and amino acid composition of toxin l’, the structure of toxin 1’ can be proposed as 3. The molecular weight of toxin 3 is 1014, and its amino acid composition is Ala, Leu, Asp, Glu, Mdha, Adda, and an unknown amino acid, but its structure remains unsolved because of the small amount isolated. In summary, we found that A. flos-aquae NRC 525-17 contains four hepatotoxins in addition to the neurotoxin anatoxin-a(s). They are microcystin-related compounds, in which two contain a rare amino acid, homotyrosine. Although Al-Lay1 et al. reported that an Anabaena produces both anatoxin-a and mirocystin (%), this is the first case to our knowledge that Anabaena produces simultaneously anatoxin-a(s) and microcystins.
Acknowledgment. We are grateful to Drs. Hisao Oka and Yoshitomo Ikai for assistance in the measurement of GC/MS and to Tsuyoshi Mayumi for amino acid analysis.
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Structure-Activity Relationships of Bacterial Mutagens Related to 3-Chloro-4-(dichioromethyl)-5-hydroxy-2(5H)-furanone: An Emphasis on the Effect of Stepwise Removal of Chlorine from the Dichloromethyl Group Robert T. LaLonde,* Gary P. Cook, Hannu Perakyla, and Lin Bu Department of Chemistry, College of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210 Received March 29, 1991
The Salmonella typhimurium (TA100)mutagenicities of six structural analogues of 3chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) were determined and compared. These were also compared to previously determined mutagenicities for another four analogues. This study was conducted for the primary purpose of ascertaining the effect of C-6 chlorineby-hydrogen replacement on mutagenicity. The compounds assayed were 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (3), 3-chloro-4-(chloromethyl)-2(5H)-furanone (4), 3chloro-4-methyl-5-hydroxy-2(5H)-furanone(7), 3-chloro-4-methyl-2(5H)-furanone(a), 4methyl-5-hydroxy-2(5H)-furanone(9), and 4-methyl-2(5H)-furanone (10). Compounds 3,4, and 7 were mutagenic whereas 8-10 were not. All six compounds were stable under assay conditions. Mutagenicity data for the three active compounds were combined with data of another four active compounds studied previously to obtain an expanded data set. Mutagenicities of the seven compounds were compared, pairwise, in 21 comparisons and then by multiple regression analysis. On the average, chlorine-by-hydrogen replacement of a single chlorine located a t a chloromethyl group (C-6) had a markedly greater effect in reducing mutagenicity than a similar replacement at C-3 or a hydroxyl-by-hydrogen replacement a t C-5. The chlorine-by-hydrogen replacement at C-6of compound 3 resulted in the greatest mutagenicity reduction of any single replacement and amounted to a 103-fold diminished mutagenicity.
Introduction This paper concerns further structure-activity investigations of chlorinated ligno-humic products possessing the 2(5H)-furanone skeleton. Among this family of compounds is 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (1, Figure 1). MX is the most potent member of this family of direct-acting mutagens. It is a product of the chlorine bleaching stage of softwood pulping (I), results from the chlorination of humic waters (2,3),and is observed in numerous samples of chlorine-disinfected
drinking water (4-8), where its contribution to total mutagenicity ranges from 3 to 33% of the water concentrate (5). The molar mutagenicity of MX in the Ames Salmonella typhimurium (TA100) assay ranges from approximately 1000 to IOOOO rev/nmol (7,9,IO).1 The gradual, stepwise reduction of Mx mutagenicity has been effected in a series of MX analogues through a sysAbbreviations of molar mutagenicity units: rev/nmol = revertants per nanomole; rev/fimol = revertants per micromole.
0893-228x/91/2704-0540$02.50/00 1991 American Chemical Society
