DiBP, one of phthalate esters, has been used as a plasticizer to produce many common consumer products. The metabolites of such phthalate esters are released into the environment, and pose potential health risks for humans and wildlife. Since the effects of DiBP on testes still remained to be obscure, the present study focused on the testicular toxicity of DiBP, and was conducted to clarify its precise mechanism.

In ChapterI, the effects of DiBP by acute and subchronic administration on testes in prepubertal rats and mice were examined, and a recovery experiment was also carried out. Twenty-one-day-old Sprague-Dawley (SD) male rats and C57Bl/6N male mice were used for these experiments. For an acute experiment, they were once given DiBP at various concentrations (100, 300, 500, 800, or 1000 mg/kg) by oral gavage and were sacrificed under anesthesia by diethyl ether 1 day later. For a subchronic experiment, they were daily given DiBP at various concentrations (100, 300, 500, 800, or 1000mg/kg) for consecutive 7 days, and 1 day later (after the last administration), they were sacrificed as well. The results showed that DiBP increased TUNEL-positive (apoptotic) spermatogenic cells in prepubertal rats testes via acute or subchronic oral administration. Especially, the administration of DiBP at higher concentrations (>500mg/kg) elicited a severe damage in prepubertal rats testes. Thus, it is obvious that DiBP reveals a testicular toxicity, similar to other phthalate esters as previously reported. In contrast, a significant increase of apoptotic spermatogenic cells could not be observed in prepubertal mice testes, even at higher concentrations and consecutive exposure for 7 days. This discrepancy suggests that DiBP has a species-specific toxicity. For a recovery experiment, SD male rats were once given DiBP (1000mg/kg), and were sacrificed at 1 day (D1) to D8 after administration. TUNEL-positive spermatogenic cells still revealed an increase in number at D1, D2, and D5. While, they abruptly decreased, and became almost the same with those in the control at D6 and D8. In case of di(n-ethylhexyl)phthalate (DEHP), after administration, 80%~90% of DEHP is excluded from the body within 24 hr, indicating that even though DEHP is almost eliminated from the body, its effects can still persist in testes. By DEHP administration, the number of TUNEL-positive cells gradually decreased from D3 to D7, and reached the same with that in the control at D9. While, DiBP caused a significant loss of testis weight at D2 and D5, and then the testis weight returned to the normal level at D6, compared to the control. Similarly, the number of TUNEL-positive cells decreased and returned to the baseline as the control at D6. It seems that the testicular recovery in DiBP exposure is earlier than that in DEHP exposure.

In ChapterII, to clarify the mechanism of DiBP effects on prepubertal rats testes, some steroidogenic enzymes mRNA expressions, and intratesticular testosterone (ITT) concentrations in rats testes exposed to DiBP or estradiol-3-benzoate (EB) were examined. Previous studies showed that hormone controls spermatogenesis, and elevation of testicular apoptosis is due to androgen suppression and exposure to xenoestrogen. While, other studies hypothesized that induced spermatogenic cell apoptosis by exposure to phthalate esters is not caused by change in ITT level associated with steroidogenesis-related genes expression in vitro. First, to clarify whether testicular histopathological (increase of TUNEL-positive spermatogenic cells) effects elicited by DiBP in rats is attributed to mimic effects of estrogenic function, the animals were exposed to pure estrogen receptor (ER) antagonist, ICI 182,780, prior to DiBP or EB administration. The results showed that the number of apoptotic cells was not rescued by inhibiting the reaction of ER. This finding indicates that the phenomenon of increased spermatogenic cell apoptosis by exposure to DiBP might not occur via the same interaction route or molecular signal pathway with estrogen. Moreover, apoptotic spermatogenic cells showed high rate in both DiBP and EB subchronic (7 days) exposure groups, whereas, after acute (6 hr) exposure to EB, the incidence of apoptotic cells seem to be less than that in the DiBP treated group. This evidence also indicates that DiBP might elicit their adverse effects through different signal pathway from EB. Additionally, although the ITT concentration was decreased at 6 hr and 24 hr after EB administration, it was restored in the DiBP treated group at 24 hr after administration, and interestingly, the incidence of apoptosis in testes revealed a slight increase at 24 hr in the DiBP treated group. These results indicate that 1) increased number of apoptotic spermatogenic cells was not directly related with ITT concentration; and 2) EB may affect hormonal systemic circulation, but DiBP seems not to disturb the endocrine circulation. Since the ITT concentration was repressed in the DiBP and EB treated groups, steroidogenic enzymes (P450scc, P450c17, 3β-HSD and 17β-HSD) mRNA expressions were examined by using reverse transcription-polymerase chain reaction (RT-PCR). P450scc P450c17 and 17β-HSD genes expressions were decreased in both DiBP and EB treated groups, while 3β-HSD was decreased only at 6 hr in the DiBP treated groups but not in the EB treated group. Therefore, it is suggested that the effect of DiBP on testicular steroidogenesis is different from that of EB. Additionally, a number of general factors (FasL, caspase-3, vimentin) that involved in apoptotic signal pathway were examined in order to compare the effects of DiBP with anti-androgen (flutamide) or xenoestrogen (EB) by immunohistochemistry. The results showed that the expression in all apoptotic factors in the treated testicular tissues was increased compared to that in the control. It seems that testicular apoptosis induced by DiBP is through well-known apoptotic signal pathway.

In Chapter III, in vitro studies using testicular tissues and Sertoli cell cultures were carried out to clarify whether DiBP affects them directly. Several studies evaluated that phthalate esters could exert their testicular toxic effects directly on cultured cells. Moreover, it is reported that estrogen induces spermatogenic cell apoptosis in vitro. These findings indicate that increased apoptosis of spermatogenic cells may not be attributed to disruption of the hypothalamo-pituitary-testicular axis. In in vitro studies, DiBP and 17β-estradiol (E2) were dissolved in pure ethanol (99.9%), and then diluted to various required concentrations by nutrient medium. Both samples were incubated at 32℃, 5%CO2, and harvested at 3 and 12 hr after initial administration. The rate of spermatogenic cell apoptosis was determined by TUNEL analysis, and steroidogenic enzymes mRNA expressions were also examined by RT-PCR. Apoptotic spermatogenic cells were frequently encountered at 12 hr after administration compared to those in the control. The mRNA level of testicular steroidogenic genes revealed no change in all analyzed samples. Immunohistochemistry showed the disruption of vimentin filaments at high concentrations of DiBP and E2. These in vitro experiments demonstrated that DiBP and also estrogen induce spermatogenic cell apoptosis, suggesting that DiBP acts directly on testes.

In conclusion, DiBP shows a species-specific toxicity. It affects testes directly and induces spermatogenic cell apoptosis with no association with ITT concentration. This study suggests that the adverse effects of DiBP differ in several points from estrogen.

Di-iso-butyl phthalate(DiBP)はフタル酸エステル類に属し、現在、可塑剤として多種の日常製品の製造に使用されている。そのフタル酸エステルの代謝物が環境中に排出されることによる、ヒト、家畜、および野生動物への健康被害が懸念されている。DiBP曝露による精巣への影響はこれまで解明されていないことから、本研究はDiBPによる精巣毒性作用およびその作用機序を解明することを目的とした。

第一章では、単回曝露(24時間)および連続曝露(7日間)を3週齢のマウスまたはラットに施した。単回および連続曝露した精巣組織切片でTUNELアッセイを行ったところ、ラットでは、両方のDiBP曝露群でTUNEL陽性細胞数の増加が見られた。このことから、DiBPは他のフタル酸エステルと同様、精巣毒性を有することが明らかとなった。しかしながら、B6マウスでは有意な精細胞アポトーシスの増加は認められず、DiBPによる精巣への影響には種差が存在すると考えられた。

第二章では、DiBPおよびestradiol-3-benzoate (EB) 曝露による精巣内テストステロン濃度(ITT)およびステロイドホルモン合成酵素のmRNA発現量の変化を検討した。まずDiBPがエストロゲン様作用を示すか否かを検討するため、3週齢ラットにDiBPないしEBの経口曝露前に、エストロゲンレセプター(ER)インヒビターであるICI182,780を皮下投与し、その後精巣の組織学的検討を行った。その結果、精細胞のアポトーシスの減少がEB群で見られたものの、DiBP群では認められなかった。このことから、DiBPによるアポトーシスはエストロゲンとは異なる作用経路によることが示唆された。加えて、ITT濃度の有意な減少はEB曝露した6および24 時間後にともに見られたのに対し、DiBP曝露群では24時間での回復が確認された。以上の結果から、1)精細胞アポトーシスの増加はITT濃度変化と直接の関連がない、2) EBと異なり、DiBPはホルモン循環系全体に影響を与えるものではないと結論付けた。次に、DiBPまたはEB曝露によってITT濃度変化を起こす主なステロイドホルモン合成酵素(P450scc,P450c17,3β-HSD,および17β-HSD)のmRNA発現変化を測定した。その結果、P450scc,P450c17,および17β-HSDの遺伝子発現はDiBPないしEBの単回曝露群において有意に低下した。しかし、3β-HSDの発現が減少したのはDiBP群のみであった。この結果も、DiBPとEB曝露は精巣内ステロイド合成に異なる影響を与えることを示唆している。さらに、DiBP、アンドロジェン・アンタゴニストのflutamide (FM)、およびEBで、それぞれ連続曝露した精巣のアポトーシス率、ITT濃度、ステロイドホルモン合成酵素発現量、および免疫染色を比較すると、ライディッヒ細胞での3β-HSDの染色性に違いが見られたが、アポトーシス率は同レベルであったことから、三者は異なった経路でアポトーシスを誘導する可能性が考えられた。さらにアポトーシス関連因子であるFas Ligand(FasL)とCaspase-3、さらにセルトリ細胞内のvimentin filamentに対する免疫染色を、DiBP、FM、およびEB曝露群の間で比較検討したところ、FasLとCaspase-3染色性の増大とvimentinの減少が全ての曝露群において確認された。このことは、DiBP曝露による精細胞のアポトーシスは、Fas/FasL経路に従うことを示唆している。

第三章では、3週齢ラットの精巣組織および単離培養したセルトリ細胞を用いて、in vitro条件下でのDiBPの直接的な精巣への影響について検討した。組織培養試験においては、DiBPおよび17β-estradiol (E2) を3ないし12時間曝露した後、精細胞アポトーシス率の算出およびステロイドホルモン合成酵素mRNA発現の変化を測定した。その結果、アポトーシスは曝露12時間後に最も増加したものの、ステロイドホルモン合成酵素mRNAの変化は全ての曝露群において認められなかった。同じ手法で単離したセルトリ細胞を曝露した結果では、高濃度のDiBPおよびE2処理群においてvimentin filament の減少が顕著に認められた。以上のように、in vitro曝露において、DiBPは有意に精細胞のアポトーシスを引き起こしたことから、DiBPの精巣への直接的な影響が確認できた。

本研究の結論として、DiBPの精巣毒性には種差が存在し、この精巣への影響は直接的でまたITT濃度に左右されずに精細胞のアポトーシスを引き起こすと推測された。本研究で明らかにしたDiBPの精巣における影響は複数の点において、エストロゲンとの違いが認められ、よってERを介した伝達経路で精細胞のアポトーシスを誘起するものではない可能性が考えられた。これらの研究成果は、農学学術上貢献するところが少なくない。よって、審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。