This is, to our knowledge, the first study to report the effects of exposure to a biologically relevant mixture of ATR and its environmental metabolites on reproductive development in the male rat. The effective AMM concentrations utilized in this study, administered during a critical period of male reproductive tract development, are considerably lower than the current no observed adverse effect level (NOAEL) for ATR derived from previous studies that focused on male reproductive end points. Our key findings were significant treatment-related delays in PPS and increased incidence and severity of prostatitis on PND120 following a 5-day in utero exposure to the AMM. The fact that these health effects were treatment-related suggests that the AMM may be as potent as or more so than ATR alone.
The data reported here are from the male siblings of females studied by Enoch et al. [24
]. In that report, significant treatment-related increases in female offspring BW on PND4 in all AMM doses tested and on PND60 in 0.87 and 8.73 mg AMM exposed offspring were described. In the current study, there was no significant increase in male offspring BW on PND4, PND21, or PND120, but BW was significantly greater than controls in the 0.87 mg AMM and 8.73 mg AMM treatment groups on PND180. Thus, in contrast to ATR alone, this low dose AMM not only had no deleterious effect on BW, but BW was in fact elevated in both male and female offspring at the same doses, though there were variations in the rate of the response across gender.
In the present study, there was a significant, albeit marginal, delay in PPS in males of the two highest AMM treatment groups, and a nearly significant delay (p
<0.06) in males of the lowest AMM treatment group. While the biological significance of a less than one day delay in PPS is not clear, these data, in accordance with the Tier 1 Screening Battery of the U.S. EPA Endocrine Disruptor Screening Program Test Guidelines [32
], indicate that PPS is clearly a particularly sensitive endpoint following developmental exposure to ATR and its metabolites and that an AMM treatment effect does exist at low levels. Further, the observation of pubertal delays in the absence of any significant reductions in peripubertal BW are consistent with studies reviewed in Cooper et al. [4
] where both male and female pubertal onset was delayed while BW was increased following early-life exposure to ATR or its individual metabolites. Data in the present study, Enoch et al. [24
], and of those described in Cooper et al. [4
] suggest that significant effects on pubertal timing following developmental exposures to low levels of ATR, its individual metabolites, or the AMM are not associated with peripubertal BW effects. To this point, the later life ramifications of delayed puberty and the mechanism(s) of action driving this response are not known.
In addition to delayed puberty, treatment-dependent alterations in the weights of other reproductive tissues have been reported in male rats following prenatal [14
] and peripubertal [33
] exposure to ATR and following peripubertal exposure to individual ATR metabolites [23
]. However, whereas puberty was mildly delayed by biologically relevant levels of the metabolite mixture in the present study, there were no persistent effects on reproductive tract or other tissue weights, a characteristic that has been observed in at least one other study which utilized similar doses of ATR (0 to 100 mg/kg/d) and a similar dosing period (GD 14-parturition) [34
]. While PPS, normally androgen dependent, was delayed in the Rosenberg study, there were no concomitant changes in intratesticular testosterone (iT) concentrations. A similar effect was also observed in the present study in which there were no significant changes in sT in males of the AMM treatment groups on PND120 and only in males in the highest AMM treatment group was sT reduced on PND180. Interestingly, in males of the 100 mg ATR treatment group, where PPS was delayed at the p
<0.01 level, sT was actually elevated on PND120. However, in that treatment group, no T-regulated tissue weights were altered; only mean AP weight was significantly greater than that of controls. Trentacoste et al. [33
] reported decreased SV and VP weights in Sprague-Dawley rats exposed to 100 mg ATR/kg BW peripubertally, but also reported decreased iT and sT, a correlation not observed in the present study. These differences may be due entirely to the timing of exposure, as the animals in the Trentacoste study were dosed peripubertally and not gestationally, as were those in the present study. While the absence of persistent effects on male reproductive tract tissue weights in the present study is certainly understandable considering the lack of changes in circulating hormone concentrations, there does not appear to be a relationship between AMM or ATR-induced pubertal delays and changes in male reproductive tract tissue weights or circulating hormone concentrations.
It is well known that hormonal changes can influence prostate development and a number of studies indicate that PRL in particular stimulates prostatic growth in rats [35
]. Although there were significant treatment-related effects on the incidence of prostate inflammation and severity in the current study, there were no concomitant changes in circulating hormone concentrations that would suggest a mode of action for prostate effects. In addition to hormonal changes, timing of exposure can also impart late-life prostate effects and a number of studies have described a critical window for male reproductive tract development following exposure to various environmental contaminants [39
]. The window of sensitivity for prostate development occurs on GD14-19, when androgen receptors are activated and the testes begin producing androgens [41
] and the animals utilized in the present study were exposed to the AMM on GD15-19. Atrazine has been shown to have an anti-androgenic effect through suppression of the conversion of testosterone to 5α-dihyrdrotestosterone (DHT) in the hypothalamus, anterior pituitary, and prostate [43
] and that 5α-DHT prostate receptors are strongly inhibited in offspring of dams exposed to ATR and DEA during pregnancy [44
]. In addition, Babić-Gojmerac et al. [45
] demonstrated that DEA, a component of the AMM, inhibited 5α-reductase at the same rate as ATR in vivo
and at a greater rate than ATR in vitro
. Though DEA is likely less toxic due to rapid metabolism and biodegradation, the metabolic product, DACT, is also a component of the AMM and has been to shown have developmental effects similar to that of ATR [22
]. These observations suggest that GD15-19 is an effective window for AMM exposure to elicit prostate effects and that other hormones, in addition to those measured in the present study, be investigated for a potential role in a mode of action for prostate effects. Furthermore, we suggest that hormones which may be affecting these developmental outcomes should be evaluated during earlier stages of life, as persistent effects have been absent in both of our studies [present, 14].
An elevated incidence of lipomatous nodules was indicated in AMM/ATR-exposed rats. Similar fat nodules have been commonly observed in dogs, horses, and humans. The pale prostatic foci observed at necropsy on the outer edges of the tissue indicated focal regions of inflammation. While there is an evident AMM-related effect on the incidence and severity of inflammation in the prostate on PND120, this was not the case over time as these effects had dissipated by PND180. It is possible that the appropriate time point necessary to observe related changes pertinent to the inflammatory effects was not assessed. In a preliminary study, PND120 males in the 8.73 mg AMM treatment group, which had the most severe prostate effects, exhibited the highest concentrations of serum IL-1α and IL-6 (data not shown), two interleukins known to have roles in the resolution of inflammation [46
]. Cowin et al. [41
] also observed increased activation of nuclear factor-kappa B-dependent genes, including IL-1α and IL-6, in prostates of Sprague-Dawley rats exposed in utero
on GD14-19 to vinclozolin. This would suggest that the prostate lesions could have been resolving or under repair and it is possible that had necropsy been conducted earlier than PND120 in the present study, the severity might have been greater, a more prominent treatment effect would have been discerned, and/or exposure-related effects (changes in hormones or tissue weight) would have been detected. Further, this study has added to the knowledge base the fact that low dose, biologically relevant exposures to ATR and its metabolites induce prostate inflammation following early life, acute exposures and this study and others [12
] confirm that these lesions show no signs of progression to neoplasm at the time points evaluated and in a strain of rat that is fairly resistant to tumor formation.
The present study, using the lowest effective concentrations of AMM, suggests that late prenatal exposure to a mixture of atrazine and its metabolites significantly delays puberty (albeit modest compared to high dose ATR) and can affect adult prostate incidence and severity of inflammation in Long-Evans rats. The low doses and short duration of exposure may be the reason that typical dose-related increases in many of the responses were limited and these findings may be exacerbated by extended exposure. However, the fact that statistically significant low-dose responses were evident following prenatal exposure to metabolites of chlorotriazine herbicides is noteworthy as these low dose effects may stem from the late prenatal time of exposure, during which growth and development of male reproductive organs are particularly sensitive to environmental agents. The importance of these findings on late life health effects is not known, as no report to date has extended past those reported in Rayner et al. [14
] or herein, but evidence in this study supports the notion that time points earlier than 120d may be more important than those in late life with respect to a mechanism of AMM effect on male reproductive development. The effects reported here occurred with no decrease in body weight at puberty, as has been previously reported for ATR alone [13
] and puberty was significantly delayed at 0.87 mg AMM which is lower than the current NOAEL for ATR or DACT (6.25 mg/kg/day total dose [28
]). Additionally, effects were consistently observed in the AMM treatment groups, where the highest effective concentration (8.73 mg AMM/kg BW) contains only 1.79 mg ATR/kg BW. These studies suggest that low dose exposures to the chlorinated metabolites of ATR, as a mixture, may elicit effects that are relevant to human health, in a manner similar to high dose ATR exposure in male rats exposed just prior to birth.