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Testicular germ cell tumor (TGCT) incidence increased steadily in recent decades, but causes remain elusive. Germ cell function may be influenced by cannabinoids, and two prior epidemiologic studies report that use of marijuana may be associated with non-seminomatous TGCT. Here we evaluate the relationship between TGCTs and exposure to marijuana and other recreational drugs using a population-based case-control study.
163 TGCT cases diagnosed in Los Angeles County from December 1986 to April 1991 were enrolled with 292 controls matched on age, race/ethnicity and neighborhood. Participants were asked about drug use by structured in-person interview. Odds ratios and 95% confidence intervals were estimated using conditional logistic regression, adjusted for history of cryptorchidism; education; religiosity; and reported use of marijuana, cocaine and amyl nitrite.
Compared to never use, ever use of marijuana had two-fold increased risk (OR=1.94, 95%CI: 1.02–3.68) while ever use of cocaine was negatively associated with TGCT (OR=0.54, 95%CI: 0.32–0.91). Stratification on tumor histology revealed a specific association of marijuana use with non-seminoma and mixed histology tumors (OR=2.42, 95%CI: 1.08–5.42).
We observed a specific association of marijuana use with risk of non-seminoma and mixed tumors. This is the first report of a negative association between cocaine use and TGCT risk. Results warrant mechanistic studies of marijuana’s effect on the endocannabinoid system and TGCT risk, and caution that recreational and therapeutic cannabinoids by young men may confer malignant potential to testicular germ cells.
Testicular germ cell tumors (TGCT) are the most common neoplasm of men 15–45 years of age1, and young survivors face significant sequelae including elevated rates of cardiovascular disease and second primary malignancies2. Understanding etiology of TGCT as the basis of prevention is therefore an important research priority. TGCT incidence has been rising for decades3, which implies a change in exposure to one or more non-genetic risk or protective factors. However, no such characteristics have been identified despite years of investigation. An established risk factor for TGCTs is cryptorchidism, or undescended testicles, which in rodent models can be caused by exogenous estrogens during key periods of development4,5. These findings, along with epidemiologic data showing the onset of greatest TGCT risk around the time of puberty, implicate aberrant effects of steroid hormones in the perinatal and peripubertal periods. Potential exposures in the perinatal period include elevated maternal estrogens and exposure to exogenous hormones such as diethylstilbestrol. However, studies of these exposures are limited by the need to retrospectively measure relevant exposures following TGCT diagnosis, 15 or more years later. Additional epidemiologic data are consistent with the possibility that risk may be related to variation in endogenous hormone levels during puberty, as proxies such as severe acne and male pattern baldness were associated with increased likelihood of testis cancer6.
More recently, exogenous compounds with postulated or demonstrated endocrine function have been proposed as possible testicular carcinogens, with polychlorinated biphenyls, organochlorines, and other persistent organic pollutants implicated by recent epidemiologic studies 7–11. Constituents of marijuana smoke may also act on the endocrine system, and marijuana use has increased in recent decades12–13 and is highest amongst men aged 15 to 20 years old in the United States13. Daling et al. therefore postulated that marijuana use may increase TGCT risk, and in a population-based case-control study14 found that TGCT cases were more likely to have smoked marijuana, risk increasing with frequency and duration of use, as well as earlier initiation of use and use ongoing until diagnosis. The association was limited to non-seminoma histologic types. Subsequent results of a smaller hospital-based friend-control study also reported an association of marijuana use with risk of non-seminoma15. Results of these studies and biological plausibility of endocrine action of marijuana constituents indicate that marijuana smoke may contain a testicular carcinogen. Alternatively, the reported marijuana-testis cancer association may arise at least in part from a tendency of men who smoke marijuana to more frequently experience unrecognized testis cancer risk factors, such as psychosocial correlates of marijuana use which may include exposure to other recreational drugs. In the present population-based case-control study, we estimated associations between testis cancer risk and exposure to marijuana accounting for several demographic factors and detailed history of use of other recreational drugs.
A population-based case-control study was conducted in Los Angeles County, California to investigate associations of testis cancer risk with exposures of the perinatal period, childhood, adolescence and early adulthood. Eligible cases were men diagnosed with TGCT between December 20, 1986 and April 4, 1991 in Los Angeles County, who were 18–35 years of age at diagnosis, spoke English, and were born in either the United States, Europe, Canada, or the Middle East. The Los Angeles County Cancer Surveillance Program identified 201 men meeting these criteria. Of these, eight had either died before being contacted or were too ill to participate, six had cognitive difficulties precluding participation, 14 could not be located, two were not invited to participate because health care providers denied permission to contact, and eight chose not to participate. The remaining 163 eligible cases (81%) participated in the study. For each participating case, we attempted to enroll up to four unaffected control men who were matched to the case on date of birth (within three years), race, ethnicity, and neighborhood of residence at the time of diagnosis. An established procedure was used to identify potential controls, by defining a sequence of housing units on specified block; attempting to identify sex, age, race and ethnicity of inhabitants of each unit; and thereby determining whether one or more appropriately matched potential controls were identified. If no potential control was found after canvassing 150 units, race was excluded from the matching criteria; and if a matched control based on the relaxed criteria was not found within 150 subsequent units, the case was not included in matched case-control analyses. The procedure described above identified 371 individuals as potential controls for the 163 eligible cases. Of these, 292 (78.7%) chose to participate. We enrolled controls that were not matched on race/ethnicity for 24 cases, and were unable to enroll any matched control for 24 additional cases. One-to-four matched controls were enrolled for each remaining case: one control for 16 cases, two controls for 95 cases, three controls for 26 cases, and four controls for two cases.
Interviews were conducted in person in participants’ homes, administered by trained interviewers using structured questionnaires. All participants were interviewed between October 16, 1987 and December 15, 1994. Information was requested for a reference period up to one year before diagnosis of TGCT for cases and the same date for each case’s matched controls. Participants were asked to provide demographic information; family history of specific cancers and urogenital conditions; personal history of some infectious diseases; and personal use of tobacco, alcohol, and numerous recreational drugs. Participants were asked if they ever used each drug, and ever users were asked the first and last years of use, and average number of times per week of use.
Relevant variables from the questionnaire were tested individually using a univariate conditional logistic regression that estimated odds ratios (ORs) and 95% confidence intervals. The present analyses were focused on use of recreational drugs in relation to TGCT risk. We selected for model-building variables that were either notable for the magnitude of their disease association or statistical significance, or were demonstrated TGCT risk factors.
All drugs were treated as dichotomous variables indicating ever versus never use, with ever use of most drugs further divided into current use at reference date or former use. Frequency of use was categorized as never, less than once per week, or more than once per week. Duration of use was categorized as never, less than 10 years of use, and 10 or more years of use. Sensitivity analyses were conducted to explore the effect of using different cutpoints for categories of marijuana frequency and duration. Duration of alcohol consumption was not queried. Due to sparse numbers, duration of mushroom use was categorized as never, below, or above 2 years (the median duration of mushroom use), and frequency and duration of use were not examined for mushrooms; barbiturates; heroin; lisergic acid diethylamide (LSD); quaaludes; phencyclidine (PCP); ethyl chloride; butyl nitrate; poppers other than amyl nitrite, ethyl chloride or butyl nitrate; or recreational drugs other than those queried in the interview.
Covariates used in multivariate models were selected by adding variables one at a time to the model and assessing whether point estimate of the odds ratio (OR) changed by more than 10%; covariates with this influence were retained in the model. The following covariates were found to be confounders of associations between use of marijuana or cocaine and TGCT: cryptorchidism (yes vs. no), religiosity (any religious affiliation vs. none), education level (college degree or higher, some college, high school diploma, or less than high school diploma). In addition, estimates of association for each other drug were adjusted for ever use of marijuana, cocaine and amyl nitrite.
Using information from original pathology reports histologic type was coded as either seminoma (International Classification of Disease [ICDO] histology codes 9061, 9062, 9063), non-seminoma (9065, 9070, 9071, 9080, 9082, 9083, 9084, 9100), or mixed germ cell tumors (GCT) (9081, 9085, 9101). For statistical analyses we distinguished between pure seminoma of any type (“seminoma”) and non-seminoma or mixed GCT of any type (“non-seminoma/mixed GCT”). These definitions follow the convention of grouping histologic types according to degree of differentiation, while constituting subgroups likely to be uniformly scored between clinical centers, and from the study enrollment period forward in time. Moreover, they are comparable with definitions used in published studies addressing marijuana use in relation to TGCT risk14,15. Heterogeneity of disease associations of selected drugs across histological subtypes of TGCT (seminoma versus non-seminoma/mixed GCT) was tested by including an interaction term between histology and the respective main variable into the conditional logistic model, together with confounders. Controls were coded with the same histology as their matched case. Histology-specific odds ratios were computed using the full model with the interaction term.
Cases were more likely than controls to have a history of cryptorchidism (10.4% vs. 2.7%) and a higher education level, and were less likely than controls to report a religious affiliation (Table 1).
Ever use of marijuana was associated with nearly two-fold increased risk of TGCT of any histologic type (OR=1.94, 95%CI: 1.02–3.68), after adjusting for education, religiosity, history of cryptorchidism, ever use of cocaine, and ever use of amyl nitrite. Compared with never users, current marijuana users had a non-significant increase in risk (OR=1.38, 95%CI: 0.67–2.87), while former users had greater than two-fold risk (OR=2.28, 95%CI: 1.17–4.43). These associations did not follow simple dose-response patterns over categories defined by duration or frequency of use. For example, compared to never users, those who reported using marijuana less often than once per week were more than twice as likely to develop TGCT (OR=2.10, 95%CI: 1.09–4.03), while those who reported more frequent use had a lesser and non-significant increase in risk (OR=1.53, 95%CI: 0.73–3.24). Men reporting less than ten years of marijuana use were more than twice as likely to develop TGCT (OR=2.09, 95%CI: 1.09–3.98), while a lesser and non-significant increase was observed for those reporting 10 or more years of use (OR=1.51, 95%CI: 0.66–3.47) (Table 2).
Men who reported ever using cocaine were found to have notably reduced risk of TGCT in analyses adjusted for above-mentioned covariates and marijuana use (OR=0.54, 95%CI:0.32–0.91). There was no indication that risk differed further across categories of frequency or duration.
Although the estimate of association between amyl nitrite use and TGCT did not achieve statistical significance (OR=0.50, 95%CI: 0.25–1.02), ever use of this drug was found to confound estimates of disease associations with ever use of both marijuana and cocaine, and was therefore included as a covariate in final analytic models. Neither pronounced disease associations nor confounding effects were noted for reported use of inhaled ethyl chloride, butyl nitrate or other poppers; hallucinogenic mushrooms; amphetamines; barbiturates; LSD; quaaludes; PCP; heroin; reported use of any recreational drug not specifically queried in the interview; or a derived variable combining reported use of any of the aforementioned drugs, alcohol, or tobacco smoked in cigarettes.
Ever use of marijuana appeared to be unassociated with risk of seminoma (OR=1.07, 95%CI: 0.37–3.07, Table 3), but was associated with a greater than two-fold risk of either non-seminoma or mixed GCT (OR=2.42, 95%CI: 1.08–5.42). Associations with non-seminoma or mixed GCT were more pronounced for former use (OR=3.04, 95%CI: 1.29–7.19), than for current use (OR=1.61, 95%CI: 0.64–4.01). However, the latter association did not achieve statistical significance, in part because there were fewer current users. Further stratification of current users’ data on frequency or duration of use yielded estimates that also were not statistically significant. Among former users there was a three-fold association of marijuana use among those reporting use less frequent than once per week (OR=3.30, 95%CI: 1.34–8.09), although the association estimated among more frequent users was somewhat lower, it did not achieve statistical significance (OR=2.21, 95%CI: 0.74–6.61). Among former users estimates within both substrata of duration achieved significance, with the association corresponding to longer duration of use (OR=6.67, 95%CI: 1.37–32.5) exceeding that for shorter duration (OR=2.81, 95%CI: 1.15–6.86). By contrast, the data suggest that any inverse association of ever using cocaine varies little between seminoma (OR=0.46, 95%CI: 0.20–1.03) and non-seminoma or mixed GCT (OR=0.63, 95%CI: 0.33–1.21).
We report a two-fold increased risk of TGCTs amongst ever users of marijuana, compared to never users. Furthermore, we observed ever users of marijuana to have a greater than three-fold risk of non-seminoma, compared with never users. In contrast, marijuana use was not associated with risk of seminoma. The moderate but non-significant elevation of risk of mixed germ cell tumors among marijuana users may represent a mixture of effects on seminoma and non-seminoma. These results accord with the general findings of both previous epidemiologic studies exploring marijuana use and TGCT risk14–15. However, the greater TGCT risk we observed among former users of marijuana compared with current users, and greater risk among infrequent users compared with frequent users was not observed in previous studies. We also observed greater risk among men who had less than 10 years of use than among those reporting use of longer duration, a pattern reported by one previous study14 when all tumor histologies were combined. Varying results may have arisen from combined influences of differing measures of frequency and duration of marijuana use employed in the three studies14–15, differing analysis models used in each, imperfect measurement of covariates, or simply from sampling variation -- since relatively small numbers of non-seminoma cases participated in each study. Differences may also have arisen in part from nonsynchronous assessment of exposure, since trends of marijuana use have been changing12,13, and enrollment into the present study tended to occur earlier than enrollment into the others. Nonetheless, results presented here confirm both the epidemiologic association of marijuana use with TGCT risk, and the specificity of this risk factor for non-seminomatous tumors.
The main psychoactive compound in marijuana, Δ9-Tetrahydrocannabinol (THC), binds and partially agonizes the human cannabinoid (CB) receptors, CB1 and CB2. These receptors are activated by endocannabinoids (ECs), mainly N-arachidonoylethanolamide (AEA), and 2-arachidonoylglycerol (2-AG). AEA also binds and stimulates the transient potential vanilloid receptor type 1 (TRPV1), which has been proposed to confer heat resistance to germ cells and maturing sperm16. AEA is degraded in the cell by fatty acid amid hydrolase (FAAH)17. CB receptors and their endocannabinoid ligands are expressed in the pituitary and hypothalamus, and have been shown to be involved in regulation of reproduction at the hypothalamic and pituitary levels17. In males, as reviewed by Pagotto18, cannabinoids have been shown to decrease luteinizing hormone (LH), to decrease testosterone production and secretion, and to suppress spermatogenesis, in accordance with reports showing reduced testosterone levels following marijuana use. Both CB1 and CB2 are also expressed in the male reproductive system, with distinct patterns of cell and developmental specificity. In mice, mRNA and protein encoded by the CB2 ortholog are expressed in all stages of spermatogenesis, while 2-AG levels are highest in spermatogonia -- germline stem cells in the sexually mature male -- and gradually diminish in subsequent stages of spermatogenesis, whilst AEA levels are relatively unchanged19. These precise expression patterns may reflect regulation of crucial events in spermatogenesis by endogenous cannabinoid signaling, since mice engineered to be unable to express the CB2 ortholog have pronounced reproductive deficits, and ECS signaling in the male reproductive system is conserved across several species19. It therefore seems plausible that unscheduled signaling by exogenous THC could impair testicular health, by disrupting the ECS either in the pituitary and hypothalamus, or in the gonad.
Cannibinoid compounds including THC have been found in various model systems to have proapototic, antiproliferative, antiangiogenic, antimigrative, and antiadhesive properties 20. Limited mechanistic investigation has shown these effects to be mediated by numerous pathways, some involving signaling through CB1, CB2 and TRPV120. The cannabinoid system is therefore increasingly investigated as a potential source of novel chemotherapeutics. Although these observations may seem at odds with carcinogenic effects of marijuana on the testis, the direction of some biological effects of cannibinoids appear to be tissue- and cell type-specific20, and we are unaware of pharmacologic research addressing cannibinoid signaling in the testis. It thus remains early to speculate whether this avenue of research will provide biological insight into the plausibility of a causal role for marijuana use in development of non-seminomatous and mixed histology tumors of the testis.
The specific association between marijuana use and risk of non-seminomatous and mixed histology TGCT has yet to be explained. However, a mechanism involving androgen signaling or response may be plausible, since THC has recently been linked to androgen levels, and based on recent results presented elsewhere, we postulate that variation in androgen receptor biology may distinguish TGCT histology. In a separate population-based epidemiologic study we found shorter CAG repeat lengths in exon 1 of the androgen receptor gene, predicted to result in greater androgen receptor (AR) transactivation21, to be associated with risk of seminoma, with the opposite association observed for non-seminoma22. However, data on the association of TGCT histology with genotypes defined by this polymorphism are not entirely consistent. Although four hospital-based case-control studies23–26 also reported an association between longer AR CAG repeats and non-seminoma, results were not statistically significant, and one of these studies found significantly greater frequency of shorter repeats in seminoma cases than non-seminoma cases24. No such difference was reported in two of these studies23,25; while the fourth reported no association between repeat length and non-seminoma alone or all TGCT, it did not report on comparisons of repeat length among those with seminoma alone with either unaffected controls or men with non-seminoma alone26. Apart from age at diagnosis, few other factors have been found to be differentially associated with seminomas and non-seminomas. Therefore, to provide further insight regarding the histology-specific association of marijuana use observed here and reported in the earlier studies14,15 it seems advisable that future TGCT research address marijuana jointly with indicators of androgen action.
This is, to our knowledge, the first epidemiologic study to investigate use of cocaine and additional recreational drugs in relation to TGCT risk. We observed an association between reported use of cocaine and lesser risk of TGCT, which appeared strongest for mixed germ cell tumors. A mechanism whereby cocaine may reduce TGCT risk has not been proposed, but a parsimonious explanation may be loss of germ cells through a cocaine-mediated process. Effects of cocaine on the murine testis suggest several means whereby exposure could plausibly result in germ cell death. Experimental administration of cocaine causes several morphological changes of the rat testis: seminiferous tubule diameter and germinal epithelium are reduced27–28, and spermatogenesis is rapidly disrupted accompanied by cell sloughing and reduction in testicular volume28. Ultra-structural changes were observed in Sertoli cells and germ cells but not in Leydig cells, suggesting germinal lines as primary site of cocaine’s testicular toxicity28. Although the mechanism underlying these changes has not been identified, Li et al.29 identified a receptor protein in rat testes that saturably and specifically binds cocaine. If this receptor is involved in testicular maintenance, the endogenous ligand may be unable to fulfill its normal function in the presence of cocaine. An equally plausible explanation, since cocaine causes severe vasoconstriction, may simply be hypoxic cell death within the testis29. Finally, a role for cocaine in opposing effects of THC was suggested by metabolic studies assessing effects of both drugs in the rat30, which identified a general trend of reduced utilization of glucose following administration of THC, which was counteracted when cocaine was administered with THC. Without suggesting a specific metabolic mechanism whereby cocaine may protect against TGCT, these results indicate interplay between the effects of these two drugs, which may be germane to the opposing associations with TGCT risk reported here.
Cocaine use was found to be an important confounder in our analyses of marijuana effects, as might be anticipated, since marijuana use is associated with a tendency to use other recreational drugs. It appears that other studies estimating associations between marijuana use and TGCT risk did not address potential confounding effects of cocaine. True effects of marijuana use in these source populations may be somewhat greater than reported, if cocaine is also associated with lesser risk in these settings.
Strengths of our study include the population-based design and extensive in-person interviews querying demographic variables, risk factors, and detailed history of use of numerous recreational drugs. Statistical power of the study was adequate to examine main disease associations with respect to dichotomized levels of strong risk factors, even within subgroups of tumor histology. However, the study size was not adequate to address marijuana dose by jointly examining associations with respect to recency, frequency and duration of use. Neither were we able to examine statistical interactions between exposures, or to statistically test heterogeneity of single exposure associations across histological subtypes.
Several cautions should attend interpretation of case-control data on self-reported drug use. Previous authors14–15 noted that controls may be less motivated than cases to report use of illicit drugs, which may create spurious associations between disease risk and drug use. However, such reporting bias seems a less likely explanation for the specific association of marijuana use with non-seminoma or mixed GCT consistently observed in the present study and in previous studies14–15. Moreover, such bias would not lead to the association of cocaine use with lesser TGCT risk observed in the present study. Therefore, observed associations are unlikely to have arisen from reporting bias alone.
However, use of each of the specific drugs investigated in this study may be associated with other unmeasured exposures, behaviors, or sociodemographic factors that may have confounded the association between marijuana use and risk of non-seminomatous TGCT. Recognized correlates of marijuana use were not completely captured by our questionnaire, as these include complex psycho-social variables, including sensation-seeking behavior, peer group integration and parental or adult supervision31. Our study did query religion, a negative correlate of marijuana use that was found to be a confounder of the marijuana-TGCT association. In order for confounding by unmeasured variables to account for reported marijuana-TGCT associations, such factors would need to be associated with marijuana use in source populations for all three studies, and to be specifically associated with non-seminoma risk. Similar forms of unrecognized bias could conceivably be present in all three studies, which were similar in numerous respects; however, it seems far less likely that the specific association with a single histologic subtype would be spurious.
We conclude that marijuana use is associated with elevated risk of TGCT, especially non-seminoma or mixed histology tumors. This consistent finding across three epidemiologic studies now warrants mechanistic research investigating biological processes whereby constituents of marijuana smoke may influence testicular carcinogenesis. Moreover, the possibility that any effect of marijuana on TGCT risk may involve perturbation of the endocannabanoid system should be considered if cannabinoid agonists or antagonists are to be used as therapeutic agents, as has been proposed for numerous conditions including endocrine-related cancers and infertility19,32–33.
FUNDING: This work was supported by grants from the National Cancer Institute (CA17054, CA136967 and CA102042) U.S. Public Health Service. The collection of cancer incidence data used in this study was supported by the California Department of Health Services as part of the statewide cancer reporting program mandated by California Health and Safety Code Section 103885. Ideas and opinions expressed herein are those of the authors and endorsement by the State of California, Department of Health Services, the National Cancer Institute, and the Centers for Disease Control and Prevention or their contractors and subcontractors is not intended nor should be inferred.