1 Department of Environmental Health and 3 Department of Biostatistics, Harvard School of Public Health, 4 Nursing Program, School for Health Studies, Simmons College, 6 Vincent Memorial Obstetrics and Gynecology Service, Andrology Laboratory and In Vitro Fertilization Unit, Massachusetts General Hospital, Boston, MA, 2 Department of Bioengineering, University of Washington, Seattle, WA and 5 Centers for Disease Control and Prevention, Atlanta, GA, USA
7 To whom correspondence should be addressed at: Department of Environmental Health, Occupational Health Program, Harvard School of Public Health, Building 1 Room 1405, 665 Huntington Avenue, Boston, MA 02115, USA. Email: rhauser{at}hohp.harvard.edu
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Abstract |
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Key words: comet assay/DNA damage/exposure/insecticides
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Introduction |
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We recently found an association between urinary metabolites of carbaryl (1N) and chlorpyrifos (TCPY) and altered human semen quality (Meeker et al., 2004a). Specifically, we found statistically significant associations between 1N and decreased sperm concentration and motility, along with suggestive associations between TCPY and sperm concentration and motility. The observed associations between 1N and sperm concentration and motility were consistent with previous studies of carbaryl exposure in humans (Whorton et al., 1979
; Wyrobek et al., 1981
) and laboratory rats (Rybakova, 1966
; Shtenberg and Rybakova, 1968
; Pant et al., 1995
, 1996
). Few human and animal studies exist for comparison with our suggestive results for TCPY.
In addition to semen parameters, laboratory measures of sperm DNA damage are increasingly used to evaluate sperm function. The comet assay has been used to monitor DNA damage in a number of different cell types (Singh et al., 1988; Olive and Banath, 1996
), including human sperm (Singh and Stephens, 1998
; Duty et al., 2003
). The comet assay relies on the electrophoretic movement of DNA out of single cells, in which low molecular fragments migrate away from the cell towards the positive electrode in a pattern resembling a comet tail. It is useful as an epidemiological end-point in environmental and occupational studies since it is a measure of DNA damage, which is predictive of fertilization and embryo cleavage rates (Sun et al., 1997
).
To our knowledge, the relationship between DNA damage in human sperm and exposure specifically to carbaryl and chlorpyrifos has not been investigated. The pesticide dibromochloropropane (DBCP), a well-documented testicular toxicant (ATSDR, 1992), was found to be associated with increased DNA single strand breaks (SSBs) in rat sperm cells (Bjorge et al., 1995
). Exposure to a complex mixture of pesticides was found to be associated with increased DNA damage in blood leukocytes and lymphocytes of pesticide production workers (Garaj-Vrhovac and Zeljezic, 2000
; Grover et al., 2003
), but it was not possible to discern which pesticides were most strongly associated with DNA damage due to the non-specific nature of the exposure assessments. Numerous animal and in vitro studies have been conducted to investigate genotoxic properties of specific pesticides, and provided evidence that both carbaryl and chlorpyrifos may be associated with genotoxic effects in various cell types (Ahmed et al., 1977a
,b
; Woodruff et al., 1983
; Onfelt, 1983
, 1987
; Lieberman et al., 1998
; Rahman et al., 2002
). The objective of the present study was to explore the association between environmentally relevant levels of carbaryl and chlorpyrifos metabolites in urine and DNA damage to human sperm cells.
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Methods |
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Creatinine (CRE) concentrations are frequently used to adjust for variable urine dilution in spot samples when measuring pesticide metabolites. However, CRE adjustment may not be appropriate for compounds that undergo active tubular secretion, which includes organic compounds such as TCPY and 1N that can be conjugated by the liver in the form of glucuronides or sulphates (Boeniger et al., 1993). In addition, CRE levels may vary by gender, age, muscle mass, race, diet, activity and time of day. Therefore, adjusting urine insecticide metabolite concentrations using specific gravity (SG) may be more appropriate than CRE. Thus, in the present study, SG-adjusted metabolite concentrations were used as the primary method for dilution adjustment. However, in addition to SG-adjusted results, volume-based (unadjusted) and CRE-adjusted TCPY and 1N concentrations were also determined for comparison with exposure distributions from other studies. Samples with CRE concentrations >300 or <30 mg/dl, or with SG > 1.03 or <1.01 were considered too concentrated or too dilute, respectively, to provide valid results and were excluded from the primary analysis (Teass et al., 1998
). CRE was measured photometrically using kinetic colorimetric assay technology with a Hitachi 911 automated chemistry analyser (Roche Diagnostics, Indianapolis, IN). SG was measured using a hand-held refractometer (National Instrument Company, Inc., Baltimore, MD).
Semen samples were preserved in liquid nitrogen (196°C) within 1 h of collection and later analysed at the Harvard School of Public Health for DNA damage by the neutral comet assay. The comet assay is used widely for somatic cell types, and has been modified to de-condense chromatin in sperm cells. Spermatazoa DNA is tightly bound by protamine molecules (Haines et al., 1998), and a more extensive lysis step allows the liberation of DNA from proteins (Singh and Stephens, 1998
). Neutral conditions were used because of the abundance of alkali-sensitive sites in sperm. Alkaline test conditions can induce damage at alkali-labile sites and produce DNA strand breaks (Singh et al., 1989
). The comet assay procedure used in the present study has been described previously (Duty et al., 2003
). Briefly, 50 µl of a semenagarose mixture was embedded between two additional layers of agarose on microgel electrophoresis glass slides. Slides were then immersed in a cold lysing solution to dissolve the cell membrane and make chromatin accessible for the enzyme digestion steps. After 1 h cold lyses, slides were transferred to a solution for enzyme treatment with 10 mg/ml of RNase (Amresco, Solon, OH) and incubated at 37°C for 4 h. Slides were then transferred to a second enzyme treatment with 1 mg/ml proteinase K (Amresco) and incubated at 37°C for 18 h. The slides were placed on a horizontal slab in an electrophoretic unit, equilibrated for 20 min, and underwent electrophoresis for 1 h. DNA in the gel was then precipitated, fixed in ethanol and dried. Slides were stained and observed under a fluorescence microscope. Comet extent, tail distributed moment (TDM) and percentage DNA located in the tail (Tail%) were measured on 100 sperm in each semen sample using VisComet software (Impuls Computergestutzte Bildanalyse GmbH, Gilching, Germany). Comet extent is a measure of total comet length from the beginning of the head to the last visible pixel in the tail. Tail% is a measurement of the proportion of total DNA that is present in the tail. TDM is an integrated value that takes into account both the distance and intensity of comet fragments:
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Statistical analysis
Data analysis was performed using SAS version 8.0 (SAS Institute Inc., Cary, NC). Descriptive statistics on subject demographics were tabulated, along with the distributions of insecticide metabolite levels and comet parameters. For insecticide metabolite values below the limit of detection (LOD), corresponding to 0.25 µg/l for TCPY and 0.40 µg/l for 1N, an imputed value equal to one-half the LOD was used. Values for comet extent, TDM and Tail% represent the arithmetic mean of the 100 cells measured per subject. Comet parameters and insecticide metabolite levels were stratified by demographic categories to investigate the potential for confounding. Spearman correlation coefficients were used to determine correlations among insecticide metabolites and between comet assay parameters. The normality of the comet parameters and metabolite concentrations was assessed and appropriate transformations were performed. Comet extent and TDM were normally distributed. Tail% was not normally distributed, but because results and interpretation from analyses using both unadjusted and log-transformed Tail% were consistent, results using unadjusted Tail% are presented for ease of interpretation. The number of CHD in each subject's semen sample was not normally distributed and an arcsine transformation was used (Zar, 1984). SG-adjusted TCPY and 1N concentrations were both log-transformed.
Univariate and multivariate linear regression were used to explore relationships between comet assay parameters and SG-adjusted urinary insecticide concentrations. Inclusion of covariates was based on statistical and biological considerations (Hosmer and Lemeshow, 1989). Age was modelled as a continuous variable and abstinence time modelled as an ordinal five-category variable (
2 days, 3, 4, 5 or >6 days). Smoking status was dichotomized by current smoker versus never smoked or former smoker, and race was categorized into four groups: white, African-American, Hispanic and other. Regression coefficients were transformed into coefficients representing an interquartile range (IQR) increase in SG-adjusted metabolite level to improve the interpretability of results. A test for interaction between smoking status and urine insecticide metabolites was assessed by including an interaction term in the multivariate linear regression models.
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Results |
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The final multiple regression models are summarized in Table IV. After adjusting for age and current smoking, the strongest and most stable association was found between SG-adjusted 1N and Tail%. For an IQR increase in 1N, Tail% significantly increased by 4.13% [95% confidence interval (CI), 1.926.32]. An IQR increase in 1N resulted in a non-significant but suggestive reduction in TDM (2.18 µm; 95% CI 4.88 to 0.50) and a non-significant decrease in comet extent (0.92 µm; 95% CI 7.39 to 5.55). A significant increase in Tail% of 2.76% (95% CI 0.89 to 4.62) was also found for an IQR increase in SG-adjusted TCPY. Conversely, an IQR increase in TCPY was associated with a significant decline in TDM (coefficient=2.47 µm; 95% CI 4.71 to 0.22) and a non-significant decline in comet extent (2.72 µm; 95% CI 8.13 to 2.68). Coefficients for the relationships between the number of arcsine-transformed CHD and 1N (0.03; 95% CI 0.18 to 0.15) and TCPY (0.02; 95% CI 0.24 to 0.17) were near zero and non-significant.
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In a sensitivity analysis, the data were reanalysed after retaining the 46 men that were excluded from the primary analysis because their urine SG was outside the acceptable range. For TDM among the 260 men, adjusted coefficients for an IQR change in both SG-adjusted TCPY (2.65; 95% CI 4.61 to 0.68) and 1N (2.39; 95% CI 4.67 to 0.13) increased. A similar increase was observed when retaining the 46 men for the relationships between comet extent and TCPY (3.44; 95% CI 8.16 to 1.26) and 1N (2.17; 95% CI 7.60 to 3.25). Conversely, adjusted coefficients for Tail% were slightly attenuated for both TCPY (2.44; 95% CI 0.804.07) and 1N (3.82; 95% CI 1.985.67), but remained statistically significant. Similar results were also obtained when analyses were repeated using unadjusted (n=260) as well as CRE-adjusted (n=260 and n=243, when 17 men with CRE outside the acceptable range were excluded) urinary insecticide metabolite levels (data not shown).
To better understand the inconsistent associations among the three comet parameters and insecticide metabolite levels (i.e. metabolite levels exhibited positive associations with Tail% versus negative associations with comet extent and TDM), correlation coefficients were calculated for Tail% and either comet extent or TDM while stratifying by TDM or comet extent, respectively. Table V shows that even though there was no overall correlation between Tail% and TDM, when the data were stratified by comet extent, a strong negative association was found between Tail% and TDM. Conversely, when the data were stratified by TDM, the weak correlation between Tail% and comet extent became stronger. These findings suggest that some previously unrecognized interrelationships may exist between comet parameters.
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Discussion |
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Associations in the opposite direction for the relationships between insecticide metabolite levels and Tail% as compared with comet extent and TDM were unexpected, but may reflect the type of DNA damage associated with these compounds. Although comet extent and TDM were highly correlated, there was no association between TDM and Tail%. However, when stratified by comet extent, a strong inverse relationship between TDM and Tail% was found (Table V). A potential explanation for the TDM and Tail% association when stratified by comet extent may relate to differing types of DNA damage. Double strand breaks (DSBs) may lead to small DNA fragments that migrate large distances from the comet head but are of low intensity (contain a small amount of DNA). This in turn will have a larger influence on TDM, which is an integrative measure of distance multiplied by intensity, as compared with Tail% which is the proportion of tail to head intensity. For example, comet tails shown in Figure 1a, c, e and g consist of many small DNA fragments (probably produced by DSBs) and possess relatively high TDM values and relatively low Tail%. In contrast, SSBs may contribute to a large proportion of DNA intensity in the tail (increased Tail%), whereas TDM (dependent on both distance and intensity) may increase less. Since there are few small fragments of DNA that are far away from the comet head, TDM does not increase at the same rate as Tail%. For example, comet tails in Figure 1b, d, f and h have relatively high Tail% and low TDM. They also appear to have DNA stretching from the comet head in the proximal portion of the comet, the length of which can depend on the number of SSBs (Singh, 2000). Thus, depending on whether SSBs or DSBs are more prevalent, for a given comet extent, the cell can have either a relatively high Tail% but low TDM (SSBs predominate) or a relatively high TDM but low Tail% (DSBs predominate), hence the negative correlation within comet extent strata. As an example, comparisons of Figure 1a and b, c and d, e and f, and g and h show an inverse relationship between Tail% and TDM within comet extent strata. Based on this hypothesis, results from the present study would suggest that urinary levels of 1N and TCPY are associated with increased DNA SSBs measured as Tail%. These compounds also showed inverse associations with DSBs, suggesting competition between the two types of DNA damage. In addition, in our assay, DSBs may be more susceptible to the loss of small DNA fragments in the gel or may create fragments too small to be measured depending on the sensitivity of the imaging technique (N.P.Singh, personal communication).
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If, as hypothesized, associations between non-persistent insecticide metabolites and DNA damage are related to SSBs, it may provide information on the possible mechanisms involved. DNA SSBs can be caused by ionizing radiation, oxidizing agents and alkylating agents (Memisoglu and Samson, 2000). Carbamates and organophosphates, including carbaryl and chlorpyrifos or their metabolic intermediates, have been suspected to act as alkylating agents on DNA bases, directly or indirectly through protein alkylation (Wild, 1975
; Ahmed et al., 1977a
; Rahman et al., 2002
). Bifunctional alkylating agents used as cancer drugs (e.g. busulfan and cyclophosphamide) are known to adversely affect spermatogenesis and cause sperm DNA damage in mammals (Bucci and Meistrich, 1987
; Qiu et al., 1995). Some alkylating agents affect spermatids and sperm without morphological damage (Jackson and Craig, 1969
). This is consistent with our previous findings that 1N was significantly associated with decreased sperm concentration and motility, but not with sperm morphology (Meeker et al., 2004a
). There may also be other mechanisms contributing to DNA damage. Carbaryl efficiently reduces intracellular levels of glutathione (Soderpalm-Berndes and Onfelt, 1988
), which could lead to an increase of reactive oxygen species that contribute to the formation of DNA SSBs and/or DSBs (Saleha Banu et al., 2001
; Rahman et al., 2002
). It is also possible that carbaryl may be reacting with nitrite in the environment or diet to create nitrosocarbaryl, which is then split into aromatic and aliphatic fragments. The aliphatic fragments then bind irreversibly to DNA, forming many alkali-labile bonds and SSBs (Regan et al., 1976
). For chlorpyrifos, damage could also be initiated by phosphorylation of the DNA, as the phosphorus moiety in organophosphates may be a good substrate for nucleophilic attack (Wild, 1975
; Rahman et al., 2002
).
Biomonitoring for insecticide metabolite concentrations in urine is a commonly used indicator of internal dose integrating the various pathways of exposure (Barr et al., 1999). However, non-persistent insecticides are metabolized and excreted rapidly, so metabolite levels measured in urine reflect insecticide exposure in the previous 2448 h. Urinary insecticide metabolite levels can vary considerably over time, and it has been suggested that a single sample may not be a reliable surrogate for long-term exposure (MacIntosh et al., 1999
). An unreliable measure would lead to increased non-differential exposure misclassification, which can attenuate associations between exposure and outcome. However, we recently found that a single urine sample may adequately predict 3 month average exposure (Meeker et al., 2004b
).
1N is a major metabolite of both carbaryl and naphthalene, and in the present study it was not possible to attribute 1N levels to either compound. Animal studies have found no associations between naphthalene and male reproductive health (ATSDR, 2003), while carbaryl has exhibited potential testicular toxicity (Rybakova, 1966
; Shtenberg and Rybakova, 1968
; Whorton et al., 1979
; Wyrobek et al., 1981; Pant et al., 1995
, 1996
). Thus, one potential limitation in using 1N as a biomarker of carbaryl is that the unknown contribution of naphthalene exposure to 1N levels may introduce random exposure misclassification and attenuate the observed associations between 1N and comet assay parameters.
Distributions of unadjusted and CRE-adjusted TCPY and 1N levels in the present study were compared with those recently reported for males in CDC's Second National Report on Human Exposure to Environmental Chemicals which was derived from samples collected as a part of the National Health and Nutrition Examination Survey (NHANES) (CDC, 2003). Median and 95th percentile unadjusted and CRE-adjusted TCPY concentrations in the present study were nearly identical to NHANES concentrations, while the present study had slightly higher median and 95th percentile concentrations for unadjusted and CRE-adjusted 1N. The similarities of insecticide metabolite concentrations to those of NHANES 19992000 suggest that exposure levels in the present study are representative of exposures to chlorpyrifos/chlorpyrifos methyl, carbaryl/naphthalene and their environmental degradates among men in the general population.
Although it is unclear currently which comet assay parameter is the most relevant measure of DNA damage in sperm and whether they reflect different mechanisms and types of DNA damage as we hypothesize, the percentage of DNA in the comet tail (Tail%) has been shown to be proportional to the frequency of DNA strand breaks (Olive et al., 1990). Also, Tail% may represent a more sensitive measure of DNA damage than both TDM and comet extent, because Tail% continues to increase with increased DNA damage while comet extent may not (McKelvey-Martin et al., 1993
). Significant associations between urinary metabolites of non-persistent insecticides and Tail% found in the present study suggest that environmental exposure to carbaryl and chlorpyrifos may be related to increased DNA damage in human sperm. However, future studies are needed to elucidate the inconsistent results between the different measures of DNA damage obtained by the neutral comet assay. Furthermore, future studies should seek to better differentiate carbaryl exposures from naphthalene exposures so that associations can be attributed unequivocally to one pesticide.
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Acknowledgements |
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References |
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Submitted on May 6, 2004; accepted on July 9, 2004.