Increased Frequency of Specific Genomic Deletions Resulting from in VitroMalathion Exposure1
Janice M. Pluth,2 Janice A. Nicklas, J. Patrick O'Neill, and Richard J. Albertini
Genetics Laboratory, University of Vermont, Burlington. Vermont 05401
ABSTRACT
Malathion is a widely used pesticide with high potential for humanexposure. Epidemiological studies suggest that individuals with chronicenvironmental exposures to pesticides have increased risks of varioushematological malignancies. The genotoxic data to date have been somewhat inconclusive with regard to malathion exposure. We have used a cellcloning assay to study the genotoxicity of in vitro exposure of human Tlymphocytes to malathion. We exposed cells in G0 to doses of malathionranging from 10 to 600 fig/ml. Mutant frequencies of treated samplesshowed both intra- and interindividual variability and, in some cases,
slight significant increases over the controls. Molecular analysis of hprtmutants resulting from both in vitro and an in vivo malathion exposurewas performed by genomic multiplex PCR. In seven in vitro experiments(using cells from four different individuals) and one experiment on anindividual exposed in vivo, one or more independent mutant(s) containinga partial deletion of exon 3 have been isolated from each individual. In fiveof the seven mutants, the deleted regions overlap extensively, revealing anarea within exon 3 exceptionally prone to deletions upon exposure tomalathion. This work provides the first evidence of an association betweenmalathion exposure and specific mutations in human T lymphocytes.Additional work is necessary to determine the underlying molecularmechanism for these deletions and how this may relate to agriculturalworkers' increased risk of cancer.
INTRODUCTION
Malathion, O,O-dimethyl-S-(l,2-dicarbethoxyethyl) dithiophos-phate (CAS No. 121-75-5), an organophosphorus pesticide, is widely
used for both domestic and commercial agricultural purposes. It isconsidered to be one of the safest organophosphate insecticides, andhas been used in large pest eradication programs in Florida, Texas,and California. Technical-grade malathion (the grade usually used foragricultural purposes) is usually 90-95% pure and may contain up to
11 impurities formed during its production and/or storage. Some ofthese impurities have been found to be significantly more toxic thanmalathion or to potentiate the toxicity of malathion (1). Malathion's
main pharmacological effect is inhibition of acetylcholinesterase enzymes leading to a build up of acetylcholine; many of the symptomsresulting from an acute exposure reflect this and include variousmuscarinic, nicotinic, and neurological symptoms. Malathion's selec
tivity is due primarily to mammals having high levels of carboxyes-
terases, which can hydrolyze malathion and malaoxon, the activemetabolite, to nontoxic intermediates that can be easily eliminated.Insects lack or have lower levels of these esterases, so are severelyaffected by malathion exposure.
Malathion has been widely studied in a variety of systems, but thereis still some controversy over malathion's mutagenic and/or genotoxic
potential for humans. No mutagenic effect has been shown in themajority of studies using bacteria and yeast (2-8). However, two gene
Received 12/7/95; accepted 3/19/96.The costs of publication of this article were defrayed in pan by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1This research is supported by American Cancer Society Grant CN-141. Department
of Energy Grant FG02 87ER60502, and Pathology Training Grant PHS I T3207122. Thecontents of this article do not necessarily reflect the policies of ACS. DOE or PHS.
2 To whom requests for reprints should be addressed, at Genetics Laboratory. 32 North
mutation studies using a mammalian system, the mouse lymphomathymidine kinase assay, and the active metabolite, malaoxon, did givepositive results (9, 10). Degraeve et al. (11) and Degraeve andMoutschen (12) found no increase in chromosome aberrations instudies using mouse bone marrow cells, spermatogonia, and primaryspermatocytes after either oral or i.p. exposure, whereas a largenumber of studies using human cells have shown a significant increase in chromosome aberrations and/or sister chromatid exchangeswith exposure to malathion (13-17). There has also been some evi
dence of malathion having a teratogenic effect; the offspring of ratsfed with 240 mg/kg/day malathion exhibited growth retardation andelevated mortality, although no negative effects were observed in theparents (18).
Many in vivo studies in which individuals were exposed to malathion and/or other pesticides have shown a genotoxic effect. Yoder etal. (19) found a 5-fold increase in chromatid breaks in farmers during
the summer season, when pesticide usage was highest. Van Bao (20)studied individuals who had been exposed only to acute doses ofmalathion and found a significant increase in the percentage of stableand unstable chromosome aberrations both immediately after exposure and 1 month later. Lipkowitz et al. (21) showed that exposure toenvironmental pesticides (one of which was malathion) resulted in a10-20-fold increased incidence of a particular cytogenetic aberration,
inversion 7 [inv (7)], in a population of agricultural workers. Althoughthis aberration itself is not oncogenic, these researchers felt that theelevated frequency of this aberration may relate to an overall increasein genomic instability, perhaps increasing the risk of cancer. Manyepidemiological studies have shown a statistically significant association between farming occupations and death from hematologicalmalignancies (22-27).
We have used a cell cloning assay to determine whether in vitroexposure of human T lymphocytes to malathion is mutagenic at thehprt locus and whether a particular molecular spectrum is induced.This assay was originally developed to study mutations occurring invivo in humans (28-33) and has since been modified for use in in vitro
studies (34, 35). The hprt clonal assay has been widely used, providing a large database with which to compare the molecular spectrum ofmutations induced by specific agents (36, 37).
MATERIALS AND METHODS
Cells and Culture Conditions
In Vitro Assay. Informed consent was obtained and peripheral bloodsamples were drawn from four male individuals, and mononuclear cells wereisolated. Equal numbers of T cells were placed in separate 25-cm2 flasks forexposed and control cultures at a density of 1 X 106/ml. A stock solution of
malathion was made in DMSO at a concentration of 500 fig/ml; furtherdilutions before addition to the culture were made in RPMI 1640. All cellculture media contained RPMI 1640 (Mediatech) supplemented with additional components for the various uses. The medium used for priming T cellsconsisted of RPMI 1640 with 10% CBS3 and 20% nutrient medium HL-1
' The abbreviations used are: CBS, calf bovine serum; CE, cloning efficiency; HPLC.
(Ventrex Laboratories, Portland. ME). TCGF was prepared as described previously (32). The medium for subculturing, plating, and growth of cells
contained RPMI 1640 supplemented with 5% CBS, 15% TCGF, 20% nutrientmedium HL-1, and 0.25 mg/ml PHA (HA- 17, Wellcome Diagnostics) and
irradiated human lymphoblastoid feeder cells. Medium used for feeding theplates consisted of 50% TCGF and 50% CBS, with 2 X 106/ml irradiated
feeder cells (and 6-TG when feeding selection plates).
In Vivo Assay. Twelve blood samples (courtesy of Dr. Vincent Garry,University of Minnesota) containing approximately 8 ml of blood each wereobtained from individuals who had been grain workers for different amounts oftime. These workers had received various pesticide exposures through theirwork, which entailed fumigation of stored grain bins. Two control individualblood samples were also obtained. All media used were essentially the same asthose in the in vitro assay, with the exception of the plating medium, which hada PHA concentration of 0.5
Mutation Induction, Expression, and Selection
The conditions for the in vitro Itprt assay were modified slightly from thosedescribed by O'Neill et al. (34). Malathion was added on day 1 to G0 cells and,
after 24 h exposure (day 2), PHA was added at a concentration of 1 mg/ml. Thecultures were incubated for 36-40 h to allow for mitogen stimulation and thenplated for CE determination in the presence and absence of 6-TG (finalconcentration, 10 p.M; ~day 3). From each flask, 2 X IO6 cells were also
subcultured in 20 ml of growth medium and cultivated further to allow formutant phenotype expression. Cells were again subcultured and plated at days5 or 6 and 7 or 8 as on day 3. Cells plated for CE were plated at I and 2cells/well without 6-TG and for calculation of MF at 1.5 x 104/well with
6-TG. Cells were fed 3-4 days after plating with 50 /xl feeder mix medium.Cell growth was scored after 10-14 days.
The in vivo hprt cloning assay was performed basically as described indetail previously (32). The mononuclear cell fractions were isolated, and cellswere counted and direct plated in 10 /J.M 6-TG for selection at 1 x IO4
cells/well and at 2 cells/well without 6-TG for CE determination. Cell growthwas scored after 10-14 days.
For both in vitro and in vivo hprt assays, CEs were calculated by the Poissonrelationship: CE = (-lnP0)/n, where P<>= fraction of negative wells and
n = average number of cells/well. The calculated MF is the ratio of the CEsin the presence and absence of 6-TG. hprt mutants were propagated in vitro,
and DNA was isolated for molecular analysis.
DNA Analysis
In Vitro hprt Assay. Southern blot hybridization was performed on 205mutant clones (from seven experiments) after Pstl and HindlU digestion with
TCR ßand y gene probes to determine clonal relationships of mutants (38).Briefly, clonal relationships are determined by the restriction patterns exhibited
after restriction with HindlU and Pstl (separately) and probing with the TCRgene probes. Clones having the same pattern in all four of the Southern blothybridizations analyzed were considered to have originated from the same celland to be replicate isolates. Conversely, clones with different rearrangementpatterns originated from different cells and, if mutant at hprt, represent independent hprt mutations. Of the mutants. 139 were determined to be independent isolates based on TCR gene analysis (55 control and 84 treated mutants).
Multiplex hprt PCR was performed as described by Gibbs et al. (39).Briefly, eight pairs of primers specific for the exons were added to a reactionmixture containing the PCR buffer [67 niM Tris-HCl (pH 8.8)-16.6 mM(NH4),SO4-5 mM 2-mercaptoethanol-6.8 /UMEDTA], 1.5 mM dNTPs, 10%DMSO, 6.7 mM MgCU, and approximately 250 ng purified DNA or 5-10 ¿¿1of crude cell extract (representing 5-10 X IO3 cells). Samples were incubatedfor 5 min at 94°Cbefore addition of Taq polymerase (Boehringer-Mannheim)and then underwent 33 cycles of the following: 94°Cfor 1 min, 59°Cfor 1 min,68°Cfor 2 min, and a final extension for 5 min at 68°C.
For mutants with alterations of fragment size on multiplex PCR amplification, the fragment was amplified using exon-specific primers, purified usingthe QIA quick-spin PCR purification kit (Qiagen), and sequenced with aninternal primer using the Taq dyedeoxy terminator cycle sequencing kit (Per-kin-Elmer Cetus, Applied Biosytems division). Extension products were elec-
trophoresed and analyzed on an Applied Biosystems 373 A Automated DNAsequencer, version 1.2.1.
In Vivo hprt Assay. Twenty mutant and 22 wild-type clones were picked
from one of the in vivo experiments with the grain workers. This individual hadbeen exposed to pesticides the longest (>5 years; primarily malathion andphosphine). These clones were expanded in vitro and molecularly analyzed, aswere the in v//ro-derived mutants.
HPLC Analysis of Maialinoli and Contaminants. Malathion was analyzed by HPLC with isocratic elution. The mobile phase consisted of 45% 0.05M potassium phosphate with 10 mM 1-hetanesulfonic acid (Sigma Chemical
Co., St. Louis. MO) adjusted to pH 6.5 with concentrated potassium hydroxideand 55% acetonitrile with 0.02% (v/v) triethylamine at a flow rate of 1.0ml/min (at ambient temperature). The HPLC system consisted of a Spectra-Physics model 8800 pump, a Kratos model 757 detector (220 nm). a Spectra-
Physics model 4200 integrator, and a Rheodyne 7125 injector. For HPLCpurity analysis, malathion samples were diluted into mobile phase, and a 20-/¿1injection volume was used. For post-column mass spectral analysis, a 200-filinjection volume was used. All analyses were performed on a Waters Nova-
Pak (3.9 x 300 mm) CI8 column.Mass spectrometry was performed with a Finnigan SSQ 7IOC single quad
ruple liquid chromatography/mass spectrometer with both electrospray and
10.00-
1.00-
Fig. 1. Cytotoxicity of malathion on human T cells over a rangeof doses. Cytotoxicity is represented as the relative CE as comparedto the controls and is graphed on a log scale. Each Cytotoxicity valuefrom the same blood sample is represented with the same symbol(•.donor 1: A. donor 2; •¿�donor 3; V. donor 4).
Fig. 2. Average induced MFs over a range of maialinoli doses.MF values for days 5-8 are averaged for all four blood samples.Columns, average MF for each dose; bars, SD. The 50-, 450-, and600-fig/ml doses are significantly elevated from the averagedcontrol value (P < 0.05).
0 30 50 80 450
Dose ug/ml
atmospheric pressure chemical ionization. For mass spectrometry analysis,HPLC peaks were collected, acidified with glacial acetic acid, and infused intothe ion source at 5 ¿ti/min.For electrospray ionization, the spray voltage wastypically 4.5 kV, and for atmospheric pressure chemical ionization, the vaporizer tube was kept at 200°C.Putative assignments of unknown contaminant
peaks were based on molecular weight.
RESULTS
Cytotoxicity of Malathion in Cultured Human T Lymphocytes.Peripheral lymphocytes from four male donors were isolated andexposed in G0 phase to a range of doses (10-600 /ig/ml) ofmalathion. Cytotoxicity values were calculated based on CEs fromday 3 platings (36-40 h after addition of PHA) and are graphed inFig. 1 as the CE expressed relative to the controls. No significantcorrelation between Cytotoxicity and dose of malathion was observed. The graph indicates that there may be some individualsensitivity to malathion, as the Cytotoxicity of one individual's
cells (Fig. 1, •¿�)exhibited slightly higher levels of Cytotoxicity ata number of the doses.
Mutagenicity of Malathion in Cultured Human T Lymphocytes. Preliminaryexperimentsrevealed mutationinductionby malathion to have a phenotypic expression time of 5-8 days (data not
shown), as was previously found for 137Cs-induced mutants (34).
Cells were plated on both days 5/6 and 7/8 of phenotypic expressionfor all doses used. Average MFs were calculated (from days 5-8platings) based on 3-11 independent determinations at each dosetested (30-600 /ug/ml), with the SE for each point represented with abar (see Fig. 2). Average treated MFs were slightly elevated from thecontrols but in most cases were not significantly elevated. When alltreated MFs were compared to all the control MFs, a significantincrease in MF was observed (P < 0.05). When analyzed separatelyby dose, only the 450-pig/ml dose showed a significant elevation inMF over the control, without exhibiting a large SD as was found withthe 50- and 600-(xg/ml doses. The largest increase in MF over thecontrols was approximately 10-fold (at 600 /J.g/ml,on day 7 plating),although this large induction was not repeatable, even when the sameindividual was assayed. Overall, mutant frequencies of treated samples showed both intra- and interindividual variability and, in some
cases, slight significant increases.MFs from the in vivoassays performed on the grain workers did not
exhibit a relationship between time worked and MF. In addition, nosignificant elevations in MF over the controls were seen (control MFsranged from 10.9 x 10~6 to 15.8 X 10^6, and MFs of fumigantworkers ranged from 3.3 X 10~6 to 29.6 X 10~6).
Table 1 Summary of multiplex PCR analysis results
Number and kind of alterations Percentage of Total gross structuralvisualized by multiplex PCR total mutants alterations(%)Control
mutants 3 total deletions 5.5 9/55(16.4)3exon 2 and 3 deletions5.51exon 4 and 5 deletion1.82shifts in exon 6"3.6Malathion-exposed
mutants* 3 total deletions 2.922/104(21.0)(In
vilrii doses 30-600 ug/ml) 1 exon 2deletion2
exon 2 and 3deletions7shifts in exon 3<1exon 3deletion1exon deletion4Iexon 4-9deletion1
shift in exon61exon 6-9deletion.0.9i.7.0.0.0.0.03exon 7-9 deletions 2.9
" Shifts are partial deletions of exons that are visualized on multiplex PCR as downward shifts of the band representing the exon indicated.h Included in malathion-exposed group are 20 in viva-derived mutants; no TCR gene analysis for clonality was performed on these mutants, and to be conservative, each was
Fig. 3. Exon 3 region of hprl showing the regions deleted in a number of malathion-
exposed mutants (JP33 M 13. JP34 M2 1 (sibs). and JP34 M 11 deletions exlending from bp16703 lo 16734; JP50B M25 and M26 deletions extending from bp 16703 to 16724; JP66M6 deletion extending from bp 16748 to 16768; JP74 M6, M8. and MI4 (all sibs)deletions extending from bp 16774 to 16796; and in v/vo-mutant LS309 M4 deletionextending from bp 16671 to 16750]. ..... , Deleted in mutants JP33 M13. JP34 M21(sibs), and JP34 M 11; —¿�. deleted in mutants JP50 M25 and M26; 1111. deleted in mutantJP66 M6; ---- deleted in mutants JP74 M6. M8. and M14 (all sibs); - . deleted in invivo mutant LS309 M4.
Molecular Characterization of 6-TG-resistant T-Lymphocyte
Clones. Mutants were isolated from in vitro experiments in whichsome induction in MF over the controls was observed. Molecularanalysis was performed on all mutants picked from the day 5/6 and7/8 platings, which grew to a sufficient number for Southern blotanalysis (~5 X IO6 cells). Twenty mutants were also picked from the
individual with the longest in vivo exposure and propagated in vitrofor molecular analysis. A summary of the multiplex PCR results areshown in Table 1. All in vitro numbers are based on independentmutants as determined by TCR Southern blots (data not shown).Experiments JP33, JP34. and JP35 were performed using the bloodsample of individual 1; JP50A and JP50B with the blood sample ofindividual 2; JP66 with the blood sample of individual 3; and JP74with the blood sample of individual 4. Only two changes were foundin the 20 mutants analyzed from the in vivo experiment, both downward shifts of the exon 3 fragment on the hprt Southern blot, one(LS309 M3) slightly smaller than the other (LS309 M4). But whenmultiplex PCR was performed on these two mutants, one had a partialdeletion of exon 3 (LS309 M4) and the other a total deletion of exon4 (LS309 M3). The breakpoints of the mutant LS309 M4 weredetermined and are depicted in Fig. 3. The exact breakpoints in theother mutant (LS309 M3) are still uncertain, and this mutation mayinvolve some complex rearrangement. Because no TCR analysis wasperformed on the in v/'vo-derived mutants, to be conservative, each
was counted as an independent mutation. The percentage of grossstructural alterations in the malathion-exposed group was slightly
higher than that of the control [21.0 versus 16.1%, respectively (seeTable 1)]. A significant difference was noticed among the types ofdeletions in each group. Thirty-two % (7 of 22) of the mutations fromthe malathion-exposed group showed deletions involving exon 3,
whereas the control group showed none. The frequency of this partialexon 3 deletion is significantly higher in the malathion-treated cellsthan in the in vitro control group's cells (P < 0.05; Table 2).
To better evaluate the significance of the partial exon 3 deletionmutation frequency, a larger database was compiled from publishedand unpublished multiplex PCR analysis of hprt mutations from anumber of laboratories (40).4'5 Approximately 40% of the mutants in
this large database were derived from normal non-exposed cells, with
the remainder coming from individuals with various genetic or otherdiseases (diabetics, colon cancer patients, fathers of Prader-Willi
in vitro mutagen-exposed situations (137Cs, radon, amsacrine, cis-
platinum, WOT, and plutonium). Therefore, the larger database,comprising 1654 hprt mutants, is heterogeneous. The frequency ofexon 3 partial deletions in the current 104 mutations derived frommalathion exposure is also significantly elevated from the frequencyfound in this much larger database (P < 0.0001; see Table 2). Of the1654 database mutants, only 6 showed a shift indicating a partialdeletion of exon 3. The mutants that did show the exon 3 shift werederived primarily from in vivo exposures, the Chernobyl incident (3mutants), and in vitro exposures of 137Csradiation (1 mutant) and the
etoposide amsacrine (1 mutant). Genomic sequence analysis wasperformed on the malathion-exposed partial exon 3 deletions. Fig. 3shows the breakpoints for the malathion-exposed partial exon 3 deletions, revealing a 3' region of exon 3 that seems to be exceptionally
breakpoint prone. Included in Fig. 3 is the region of exon 3 founddeleted in a mutant analyzed from an in vivo pesticide-exposed (to
malathion and phosphine. primarily) individual. The region deleted inthis in viYoderived mutant totally encompasses four of the regionsdeleted in the in v;'fro-derived partial exon 3 mutants and overlaps
slightly with the region deleted in another. In most cases, the breakpoints for the partial exon 3 deletions in the larger database of 1654hprt mutations have not yet been determined. Two, however, obtainedin our laboratory after in vitro 137Csexposure, have been sequenced.
These two independent mutations (by TCR gene analysis) have identical breakpoints (bp 16,760-16,813); the region deleted in thesemutants overlaps slightly with the deletion in one of the malathion-
induced mutants (JP66 M6; overlaps 9 bp), and totally encompassesanother (JP74 M6, M8, and M14. all sibs).
An attempt was also made to narrow down the breakpoints for ani/i vitro total exon 3 deletion mutant, isolated from the malathion-
exposed cells. A battery of PCR primers surrounding exon 3 wereused in combination to determine the approximate breakpoints for thisdeletion. On the basis of the size of the products obtained and hprtSouthern blot studies, this mutant appears to have a large insertion(>20 kb), the placement of which has been narrowed down to theregion encompassing base pairs 16.676-16,695 (within exon 3). The
breakpoints for this likely insertion lie within the region deleted in thein v/'vo-derived mutant in Fig. 3. This mutation was not counted in the
total numbers of exon 3 partial deletion mutants because it is likely aninsertion, but it is of interest in that another mutation occurs withinthis particularly malathion-sensitive region of hprt.
Determination of Malathion Purity. Each lot of malathion wasanalyzed using mass spectrometry and HPLC to determine whetherdifferent percentages of contaminants may have affected the mutage-
nicity results. The various lots appeared to be very similar with regardto the percentage and types of contaminants; they contained approximately 1-3% contaminants. There were three lots of malathion pur
chased from Supelco at various time points in the study. The first hadapproximately 2% total contaminants consisting of three primaryspecies (isomalathion, a malathion isomer analogue, and a species thatis likely malaoxon). The second had primarily two species of contaminants that made up the total 3% contamination (the malathionisomer and the malaoxon-like contaminant). The third contained the
Table 2 Frequency of emn 3 shifts as visualized by multiplex PCRin vdr/ÃÃw.vdatabases
Total Number of mutants Percentage of mutantsPopulation analyzed with exon 3 shifts with exon 3 shifts
least amount of contamination (i.e., 1%, all of which was the mala-
thion isomer). These slight differences in contaminants probably didnot affect our results. One trial run comparing lots 2 and 3 did notshow any obvious differences in MF induction or cytotoxicities withrespect to the lot used.
DISCUSSION
A wide range of malathion doses were used in the current study(i.e., 10-600 pig/ml). In extreme cases, a farmer or other agricultural
worker could occupationally experience the lower doses. The estimated lethal oral dose of malathion for humans is 858 mg/kg bodyweight. The concentration of malathion found in the blood of individuals autopsied after malathion overdose ranged from 175-517
/ug/ml (41). Therefore, concentrations considerably less than these(i.e., less than 100 jug/ml) could be achieved in the blood throughvarious nonlethal human exposures. Our studies over a range of dosesreveal malathion as having a low and variable cytotoxicity, even at thehighest dose (600 jag/ml). Also, there may be individual differences insusceptibility to the insecticide as shown in Fig. 1, with one individualshowing higher cytotoxicities over a range of doses.
The mutagenicity of malathion was also variable. Significant increases in MFs in treated versus nontreated cells could be shown onlywhen all treated MFs were averaged and compared to the nontreatedMFs (P < 0.05). A slight dose-response relationship was also seen.Others, too, have failed to find dose-related genotoxic responses with
malathion exposures but were able to detect significant differencesbetween treated and control groups (13, 15, 42). These cytogeneticstudies over a range of malathion doses failed to detect a correlationbetween the frequency of sister chromatid exchanges and/or chromosome aberrations and dose. Several in vivo studies also failed to showa dose-response relationship in mice or humans exposed to malathion(20, 43, 44). Studies examining malathion's effect on cell cycle
progression have shown it to be a very potent cell cycle inhibitor, asreflected by lowered mitotic indices and diminished levels of radio-labeled DNA and RNA (43, 45-48). Therefore, malathion's affect on
the cell cycle is likely to be partially responsible for our lack of adose-related mutagenic response.
An additional source of variability in our studies was the poor
solubility of malathion in the medium used. This could have causedunequal distributions and exposures of cells. In one study of in vitromalathion-exposed T lymphocytes that did show a relationship between sister chromatid exchanges and dose, a surfactant F-127 plu-
ronic, rather than DMSO or ethanol, was used as the initial solvent(14). However, the use of a 5% solution of F-127 pluronic did not
result in reproducibly better results for our mutagenicity studies thanthe use of DMSO, so we continued to use the latter solvent. Theability of Garry et al. (14) to show a dose-response relationship may
reflect different end points studied and/or the temporal aspects of thedifferent assays. The overall effect of the inhibition of the cell cycleby malathion and its unequal dispersion in the culture medium probably resulted in cells at the same exposure level receiving differentdoses, giving the variable and slight responses that we found.
The above complications of the in vitro mutagenicity assays makethe molecular findings of the current study even more striking. Whenmutants from the treated cultures were analyzed, a clear difference inthe molecular mutational spectrum as compared with that of untreatedcells became evident. Several mutants from the treated culturesshowed partial deletion in hprt exon 3, whereas none with this DNAchange were found any nontreated mutants. There was also specificityin the deleted region, with four of the six in vi'/ro-derived treated
mutants showing overlapping deletions in the 3' region of exon 3.
Furthermore, an additional in wVo-derived mutant had the largest
partial deletion of exon 3 (i.e., 80 bp), which encompassed the regiondeleted in four of the in vifro-derived mutants and overlapped with a
region deleted in another. The finding of seven independent mutations(at least one from each individual's in w'/ro-exposed blood sample),
represented by one or more mutants, in the malathion-treated cells
with partial exon 3 deletions and none in 55 independent in vitromutations from untreated cells, strongly supports the role of malathionin inducing these deletions.
To determine the extent to which this intra-exon 3 deletion couldserve as a "signature" of a malathion-induced genotoxic event, we
compiled a larger database of hprt mutation determinations by multiplex PCR, using published and unpublished results from severallaboratories, including our own. In a total of 1654 mutants analyzedby multiplex PCR, only 6 showed a partial deletion of exon 3 (see
Fig. 4. Hairpin-loop structures that may form in theexon 3 region of hprt. Breakpoints for each independentmalathion-induced mutant are shown, and 5' or 3' break
point is indicated |JP33 M13, JP34 M21 (sibs). and JP34Mil deletions extending from bp 16703 to 16734;JP50B M25 and M26 deletions extending from bp 16703to 16724; JP66 M6 deletion extending from bp 16748 to16768; JP74 M6. MS. and MI4 (all sibs) deletions extending from bp 16774 to 16796; and in vim mutantLS309 M4 deletion extending from bp 16671 to 16750].The number of deletion breakpoints at each of these sitesis shown in parentheses; *. in w'w-deletion breakpoints.
Table 3 Short nomologie* surrounding nialalhìan-ìnducedpartial exon 3 deletions
Mutants Region surrounding partial exon 3 deletion
LS 309 M4(in vivo derived)JP33 M13 and JP34 M21 (sibs)0
(individual 1)
JP34M11(individual I)
JP50B M25(individual 2)
JP50B M26(individual 2)
JP66 M6(individual 3)
JP74 M6, M8. and M14 (all sibs)(individual 4)
[5'region//. . .deleted region. . .//3'region]
GTGTGC//TCAAGG //ATGACGTGTGC// ATTCCT//ATGAC
GACCTG//CTGGAT //TGATAGGACCTG// AAAT AG//TGATAG
GACCTG//CTGGAT //TGATAGGACCT// AAAT AG//TGATAG
GACCTG//CTGGAT //ATAOAAGACCTG //CACTGA//ATAGAA
GACCTG/ /CTGGAT //ATAQAAGACCTG// CACTGA//ATAGAA
o*ATCCATT//CCTATG.ATCCATT//. . .G6.
. . G. ..//CAGACTTTTTATC//CAGACT
TCAGAC//TGAAGA //TTAATATCAGAC//. .GTATAT//TTAATA
" Sibs, replicate isolates of the same in vitro mutation.
This nucleotide found inserted between the deletion breakpoints.
Table 2). None of these intra-exon 3 deletions in this larger database
were from control individuals or cells; all were associated with in vivoor in vitro exposures to known clastogenic agents. Thus, the percentage (6.7%) of intra-exon 3 deletions in the malathion-exposed cells is
significantly greater than even that found in this larger database(0.4%; P < 0.0001).
Mechanistically, it is unknown how malathion produces genomicdeletions and, moreover, why they are specific and restricted to afairly small region (125 bp). An analysis of the exon 3 sequenceshows that it has the potential to form up to four hairpin-like struc
tures, each with a stem of at least 4 bp, the AG values of which rangefrom 0 to -5.8 kcal/mol. The two most stable hairpin structures are
depicted in Fig. 4 with the breakpoints for the malathion inducedmutants labeled. It is possible that malathion stabilizes these hairpin-
loop structures leading to the characteristic breakpoints. There appearsto be some association between the breakpoints and these hairpinstructures because four of the six deletion breakpoints of the inwire-derived mutant reside in the larger second hairpin structure, andthe deletion breakpoints of the in vivo-derived mutant are near the
base on either side of this structure.Small direct repeats of 2-5 bp have been associated with the
breakpoints in spontaneous deletion mutants (49-51). Although smalldirect repeats (2-5 bp) were found at or near the breakpoints in all thein vi/ra-derived malathion-associated mutants (shown in bold type in
Table 3), it is difficult to understand why they would only increase thefrequency of deletions at these sites in the treated cells. It is possiblethat malathion affects the ability of one or more DNA repair enzymesto function accurately, leading to an increased frequency of deletionsin the treated cells, but again, this explanation does not explain theincreased frequency of deletions in such a small region of hprt.
Previous studies of malathion's interaction with DNA have estab
lished only a possible weak interaction. Malathion was able to inducebreakage in Col El plasmid DNA from E. coli at a concentration of0.01 mg/ml (8). Malathion was also able to alkylate nitrobenzylpyri-
dine [a synthetic substrate (52)] and methylate DNA bases in vitro(53). However, the ability to methylate and alkylate DNA does notexplain malathion's apparent ability to cause genomic deletions,
because other alkylating agents normally induce primarily pointmutations.
The results of this study have potential health implications forhumans. First, we have shown that malathion mutagenicity can bedetected at the molecular level. The molecular changes at hprt wereseen at doses of malathion that produced no cytotoxicity in vitro (:£
50 mg/ml) and at exposure levels experienced by the individual fromwhich the in vivo mutant was obtained. Inasmuch as several epide-
miological studies have found an increase in hematological malignancies in individuals occupationally exposed to pesticides, our finding ofmalathion genotoxicity may be especially relevant. Similar changes tothose reflected in hprt in our study may be occurring also at other loci,especially sites of oncogenes or tumor suppressor genes, and may playa role in the induction of malignancies in individuals exposed to thisor a similar agent. Finally, our finding of specificity of the malathioninduced genomic deletions at hprt may have a practical relevance inallowing the development of a molecular-based assay for the rapid
detection of in vivo genotoxicity in individuals exposed to pesticides.Molecular epidemiology can then supplement traditional epidemiology in elucidating the true health effects of these potentially widespread human exposures.
ACKNOWLEDGMENTS
We gratefully thank Dr. Vincent Garry (University of Minnesota) for thegrain fumigant workers' blood samples and Drs. James C. Fuscoe of EPA
(Research Triangle Park, NC), Irene Jones (Lawrence Livermore Laboratory,Livermore, CA), Barry Finette, and Scott Clark (University of Vermont.Burlington, VT) for sharing their multiplex PCR data bases. We also thankLinda Sullivan, Tim Hunter, and Dr. James Bigelow for technical assistance.
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