We found inverse associations between urinary TCPY and testosterone levels and FAI. Increased urinary 1N concentrations were also associated with decreased testosterone levels. The concentrations of insecticide metabolites among men in our study are environmentally relevant, because the distributions of TCPY and 1N were similar to those reported in the U.S. general population.1
In secondary analyses, the association between TCPY and testosterone was not sensitive to the modeling approaches used. However, we found some differences between primary and secondary analyses. When men with extremely diluted or concentrated urine were retained in the analyses, there was a suggestive association between TCPY and LH (not found in the primary analyses that excluded men with dilute or concentrated urine). In addition, the dose–response relationship between testosterone and 1N became weaker when using exposure quintiles.
Although physiological mechanisms underlying the inverse association between TCPY and testosterone remain unclear, our understanding of the hypothalamic–pituitary–gonadal axis may provide some insights. Gonadotropin-releasing hormone (GnRH) from GnRH-secreting neuroendocrine cells in the hypothalamus stimulates gonadotrophs in the anterior pituitary to produce and release both LH and FSH. LH then acts on the Leydig cells to stimulate testosterone secretion. Through negative feedback, testosterone decreases the production of LH by acting on either the hypothalamus to decrease GnRH secretion or on the anterior pituitary to decrease LH secretion.23 Thus, if the site of action of TCPY was the Leydig cells in the testes, a positive association between TCPY and LH might be expected to accompany the inverse association between TCPY and testosterone observed in the present study. However, we found a suggestive inverse association between TCPY and LH. This suggests TCPY or its parent chemicals may be acting on the hypothalamus or anterior pituitary to disrupt LH production or secretion. Chlorpyrifos is an inhibitor of cholinesterase activity and at low doses has been found to cause a dose-dependent inhibition of cholinesterase in the hypothalamus of rats.24 Cholinesterase inhibition in the hypothalamus may in turn alter the rate of GnRH secretion.25 In addition to effects on LH, altered GnRH secretion may also affect the production and secretion of FSH in the anterior pituitary. However, the cyclic release of FSH is governed to a lesser extent than LH by varied secretion of GnRH, and feedback control of FSH secretion is less clearly defined than that of LH.23,26 We found no association between TCPY and FSH, which may suggest that TCPY alters GnRH recognition and/or LH secretion by the anterior pituitary, but not FSH secretion. In support of our hypothesis, the pituitary has been suggested as a site of action by phytoestrogens due to their ability to inhibit GnRH-induced LH release by gonadotrophs in female rats.27,28
The localization of the neuroendocrine axis, and specifically GnRH neurons, in the brain makes them anatomically and physiologically situated to mediate effects of reproductive and neurologic toxicants found in the environment.27 However, limited studies have investigated the association between exposure to chlorpyrifos/chlorpyrifos methyl or carbaryl/naphthalene and reproductive hormone levels. Several studies suggest chlorpyrifos may be hormonally active, but specific findings have not been fully consistent. In female sheep, Rawlings et al11 found an association between chlorpyrifos and thyroxine and cortisol, but no association between chlorpyrifos and LH or FSH. Andersen et al13 reported weak responses to chlorpyrifos in 2 estrogenicity assays in vitro. On the other hand, chlorpyrifos-methyl, an analog of chlorpyrifos, exhibited antiandrogenic activity, but not estrogenic or estrogen-like activity, in immature rats.14 In another study, chlorpyrifos was found to potently affect GnRH gene expression and biosynthesis in vitro.12 In addition, chlorpyrifos and other organophosphates have been shown to inhibit steroidogenesis in adrenal cells.29,30
Limited studies also suggest carbaryl may be a hormonally active agent, but similar to the studies of chlorpyrifos, specific findings lack consistency. An in vitro study of human breast and endometrial cancer cells exposed to carbamate insecticides concluded that carbaryl may act as a general endocrine modulator in mammalian cells.31 Carbaryl was associated with decreased GnRH and gonadotropic hormone levels in fish under both laboratory and field conditions.10 Conversely, carbaryl exposure among rats was associated with a dose-dependent increase in pituitary gonadotropic function.32
An alternative mechanism of hormonal effects may be directly through the phenolic metabolites (TCPY or 1N) rather than the parent compounds. These compounds may interfere with hormone signaling in the human body, although we are not aware of any studies that support or refute this hypothesized alternative mechanism.
Human studies on nonpersistent pesticide exposure and reproductive hormones are limited. A study among Danish farmers found that traditional farmers, who were presumably more highly exposed to pesticides than organic farmers, had a lower testosterone/SHBG ratio (free androgen index).33 Among Chinese factory workers exposed to the organophosphates parathion and methamidophos, Padungtod et al34 found exposure was associated with increased serum LH and decreased serum testosterone. Two animal studies found inverse associations between organophosphate exposure (diazinon or malathion) and testosterone levels that were also accompanied by a decline in semen quality (decreased sperm count and motility, increased percentage of morphologically abnormal sperm).36,37 These results are largely, although not entirely, consistent with our findings, because we found TCPY and 1N to be associated with a decline in testosterone and, in previous work, semen quality.8
The cohort of men in the present study overlap with men from our previously published studies on semen quality and DNA damage in sperm.8,9 We previously found evidence of an inverse association between TCPY with sperm concentration and motility and between 1N with sperm concentration. There was a strong and consistent inverse association between 1N and sperm motility. We also previously reported positive associations between TCPY and 1N and DNA damage in sperm cells, with 1N exhibiting a stronger association. In the present study, we found negative associations of TCPY and 1N with testosterone; TCPY demonstrated a stronger dose-dependent relationship.
There are limited human data on the relationship between testosterone and semen quality. Among normal couples, Uhler et al35 found associations between FSH and inhibin B with semen quality, but reported no associations between testosterone and sperm concentration, motility, or morphology. However, in our data, we observed an association between testosterone and sperm motility when adjusting for SHBG (unpublished results). In an attempt to synthesize the results from our 3 studies, we therefore relied primarily on studies in experimental animals. In our studies, we found associations between TCPY with testosterone and associations between 1N with semen quality and sperm DNA damage. The differences in results for TCPY and 1N may suggest differing sites or mechanisms of action by chlorpyrifos and carbaryl/naphthalene on the male reproductive system. The strong association between TCPY and testosterone suggests that chlorpyrifos may be associated with altered endocrine function of the male hypothalamic–pituitary–gonad axis; in contrast, the strong associations of 1N with sperm motility and DNA damage suggests that carbaryl or naphthalene may be associated with direct damage to developing or mature sperm.
A potential limitation of the present study was the collection of only a single urine sample to measure insecticide metabolite levels and the collection of a single blood sample to measure serum hormone levels. Despite the diurnal and pulsatile fluctuations in serum hormone levels, a single blood sample can be used to provide a reliable measure of testosterone and LH over both short and long time periods in population studies.38,39 Also, requiring multiple blood samples may limit participation rates in epidemiologic studies.38 Measuring insecticide metabolite levels in urine provides a measure of individual internal dose. However, nonpersistent insecticides are metabolized and excreted rapidly, and levels of both TCPY and 1N measured in urine reflect insecticide exposure in the previous 24 to 48 hours.40 Although insecticide metabolite levels in urine can vary considerably over time, suggesting that a single urine sample may not be a reliable surrogate for longer-term exposure,41 we recently showed that a single urine sample was predictive of 3-month average urinary insecticide metabolite levels.16 A single urine sample correctly classified men in the highest 3-month exposure tertile with a sensitivity of 0.6 and specificity of 0.9 for SG-adjusted 1N and, for SG-adjusted TCPY, a sensitivity of 0.5 and specificity of 0.8.
In our study, recruitment of subjects through an infertility clinic is not likely to introduce selection bias. A recent study among a cohort of men that overlaps with the men in the present study reported no differences when semen characteristics were compared between participants and nonparticipants, suggesting that men did not participate based on semen quality.42 Likewise, we believe it is unlikely that men participated based on their hormone status or based on exposure to nonpersistent insecticides. In addition, the participation rate in the present study (65%) was higher than other male reproductive health study designs, which reduces the potential for selection bias and increases the internal validity of a study.
The study included both fertile and infertile men, because the female partner's infertility may be the cause of some couples’ infertility and subsequent evaluation. Furthermore, pesticide metabolite levels in the present study were similar to those found in the general population, suggesting that men from the infertility clinic did not have widely different levels of exposure. For these reasons, we believe the results are generalizable to men in the general population. For generalizablity to be limited, men in the present study would need to be differentially affected by exposure (ie, more susceptible to exposure) compared with other men. There is currently no evidence suggesting that reproductive hormone levels in men visiting an infertility clinic are more sensitive to nonpersistent insecticide exposure than in other men.
In conclusion, we found an inverse association of urinary levels of metabolites of chlorpyrifos/chlorpyrifos methyl and carbaryl/naphthalene with serum testosterone. The urinary levels of these metabolites are environmentally relevant, as shown by recent NHANES data.1 We also found a suggestive association between chlorpyrifos metabolite and decreased LH. The inverse associations with testosterone are consistent with results from our previous work, in which we found inverse associations between exposure to these insecticides and sperm concentration and motility.8 As far as we are aware, the present study is the first to investigate these associations in humans, and additional studies are needed to substantiate our results.
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In the Next Issue
Coming in March (Selected papers)
The continuing enigma of pyloric stenosis
Estimating disease prevalence in a Bayesian framework
Association between physical activity and magnetic field exposure in pregnancy
Race, cardiovascular reactivity and pretermdelivery among military women
Prepregnancy BMI, weight gain during pregnancy and the risk of preterm delivery
Validation of adolescent diet recalled by adults
Mortality among augmentation mammoplasty patients: An update
C-reactive protein and cognitive function in older women
Pendimethalin exposure and cancer risk among pesticide applicators
Effect of hydrazine exposure on cancer incidence and mortality in aerospace workers
Causes and mechanisms: An interview with Jeremiah Stamler
The Changing Face of Epidemiology. Part 1. Epidemiology and the obesity epidemic
PLUS: Editorial and commentaries on the journal's policy regarding conflict of interest