Probasin-promoter-driven expression of prolactin in mouse prostate epithelium (Pb-PRL mice) results in prostatic hypertrophy exhibiting features of human BPH, including epithelium hyperplasia, increased stromal cellularity and stromal infiltration of lymphocytes and macrophages as typically observed in the human disease [1]. It is well established that oxidative stress and oxidative DNA damage are important in the pathogenesis of BPH in both men and mice [2], therefore, the objective of this study was to test the effects of an antioxidant on prostate hyperplasia and urodynamic parameters in conscious Pb-PRL mice. We used anethole trithione (ATT) recently shown to act as a specific inhibitor of reactive oxygen species (ROS) production at the IQ site (the site of superoxide/hydrogen peroxide production in complex I that is dependent of the quinone) of the mitochondrial respiratory chain [3].

Specific and Opportunistic Pathogen Free (SOPF) male C57Bl/6J mice were generated from frozen embryos and genotyped by a specialized laboratory. For urodynamic studies, mice were transferred into specialized urination cages in a separate quiet room of the animal facilities with a 12:12 hour light-dark cycle, with food and water ad libitum. After the acclimation period of 24 hours, data on voided urine (weight and timing) and water consumption (volume and timing) were continuously collected for each mouse over further 24 hours using a data acquisition system. The following parameters were quantified: food and water intake, urine output volume, voiding frequency (in times in dark versus light periods), urine volume per voiding, voiding duration and mean uroflow rate. For experiments involving chronic treatment of Pb-PRL mice with ATT or its vehicle, we determined the basal urodynamic parameters by putting mice during 48 hours in metabolic cages. Treatment with ATT (60 mg/kg/day) or its vehicle was started immediately after by oral gavage once a day for 30 consecutive days. At the end of urodynamic recordings, mice were euthanatized and the urogenital tissues were weighted on an electronic microbalance.

Validation of the experimental model. In order to validate our BPH model, we first compared urodynamic parameters in wild-type (WT) mice (n=15; aged 6 months) and Pb-PRL mice (n=17; aged 5-8 months). In both groups, values for voiding frequency (VF), urine output and water intake were significantly greater in the dark period with respect to the light period, as expected (Two-way ANOVA. P< 0.05). VF in the dark period was significantly higher in Pb-PRL mice (5.29 ± 0.47) with respect to controls (3.27 ± 0.38; two-way ANOVA. P < 0.0001; Figure 1A). No difference was noted for VF in the light period. Again in the dark period, urine volume per voiding (Figure 1B) was significantly lower in Pb-PRL mice (140.8 ± 16.6 µL) with respect to controls (280.3 ± 42.0 µL; two-way ANOVA. P= 0.0069). No difference was noted for this parameter in the light period (Figure 1B). Urinary flow rate, in the dark period only was significantly lower in Pb-PRL mice (16.2 ± 1.33 µL/s) with respect to controls (25.9 ± 3.22 µL/s) as tested by Two-way ANOVA. P < 0.05; Figure 1C).  No statistically significant differences were observed for urine output values and water intake between the 2 genotypes.
Effect of oral gavage with ATT on Pb-PRL mice.  Following determination of basal urodynamic parameter, Pb-PRL mice (aged 12 -15 months) were treated with ATT (n=11) or its vehicle (n=12) for 30 consecutive days. At sacrifice, no difference in body weight was found between Vehicle- and ATT-treated mice. A strong decrease of the prostate/mouse weight ratio was recorded in the ATT-treated group versus the Vehicle-treated group (5.53±0.44 mg/g versus 8.93±0.94 mg/g; P= 0.0056 by Mann-Whitney test). ATT treatment had no effect on the organ weight/body weight ratio for the urinary bladder and urethra. In the vehicle-treated group, VF in basal conditions was 3.4±0.7 and 7.4±0.7 in the light and dark periods, respectively, whereas, following treatment for 30 days (Figure 2A) values were 3.17±0.49 and 5.92±0.61 micturitions in the light and dark periods, respectively (P >0.05). In contrast, in the ATT-treated group, basal values for VF were 4.27±0.73 and 7.91±0.61 micturitions in light and dark periods respectively, whereas values post-treatment were 3.09±0.21 and 5.27±0.38 micturitions in light and dark periods, respectively (Figure 2A). This difference was statistically significant during the dark period (P =0.0016 by Two-way ANOVA) but not the light period (P = 0.164 by Wilcoxon test). In the vehicle-treated group, mean urine volume per voiding was 0.246±0.022 mL and 0.244±0.021 mL before and after drug treatment, respectively, whereas in the ATT-treated group, it was 0.225±0.025 mL and 0.302±0.027 mL before and after drug treatment, respectively (Figure 2B). This difference was statistically significant (P=0.0098 by Wilcoxon test). No differences following antioxidant treatment were noted on the other urodynamic parameters.

In the first step of this study, we validated Pb-PRL mice as a relevant preclinical model of BPH. Using up-to-date urination cages, we showed that VF was significantly higher in Pb-PRL mice with respect to controls, whereas urine volume per voiding and urinary flow rate were significantly lower in Pb-PRL mice with respect to controls. Therefore, we conclude that prolactin overexpression in mouse prostate epithelium can generate a phenotype of urinary symptoms which are quite similar to LUTS in human BPH.  In the second step of the study, we demonstrated that chronic treatment with an antioxidant drug induced a highly significant decrease of prostatic lobe weight, whereas no effect on urinary bladder and urethral tissue weight was observed. Moreover, we found that antioxidant treatment reduced VF compared to pre-treatment values, specifically in the dark period. In the meantime, ATT (but not its vehicle) significantly increased urine volume per voiding, suggesting that treatment could facilitate the storage function. Thus, VF decrease induced by ATT could be directly related to the increase of the urine volume per voiding.  Finally, there was a trend towards increased urinary flow rate after ATT treatment, and no effect on micturition duration as well on urine output (diuresis).

In conclusion, we have demonstrated that Pb-PRL mice exhibit all the features of human BPH including inflammatory infiltrates [1] and urodynamic alterations, therefore it could be helpful to understand the biological mechanisms driving BPH development in humans. Moreover, our results suggest that anti-oxidant drugs (here ATT) could be a promising therapy for treatment of BPH in humans, potentially showing effects on both static and dynamic components of the outflow obstruction, differently from all other pharmacological treatments on the market. Investigations on the molecular pathways modulated by ATT in mouse prostate are ongoing.

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Continence 7S1 (2023) 100967
DOI: 10.1016/j.cont.2023.100967