It is useful to identify and examine organisms that may prove useful for the treatment of dye-contaminated wastewater. Here, we report the purification and characterization of a new versatile peroxidase (VP) from the decolorizing microbe, Thanatephorus cucumeris Dec 1 (TcVP1). The purified TcVP1 after Mono P column chromatography showed a single band at 43 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequencing revealed that the N terminus of TcVP1 had the highest homology to Trametes versicolor MPG1, lignin peroxidase G (LiPG) IV, Bjerkandera adusta manganese peroxidase 1 (MnP1), and Bjerkandera sp. RBP (12 out of 14 amino acid residues, 86% identity). Mn(2+) oxidizing assay revealed that TcVP1 acted like a classical MnP at pH approximately 5, while dye-decolorizing and oxidation assays of aromatic compounds revealed that the enzyme acted like a LiP at pH approximately 3. TcVP1 showed particularly high decolorizing activity toward azo dyes. Furthermore, coapplication of TcVP1 and the dye-decolorizing peroxidase (DyP) from T. cucumeris Dec 1 was able to completely decolorize a representative anthraquinone dye, Reactive blue 5, in vitro. This decolorization proceeded sequentially; DyP decolorized Reactive blue 5 to light red-brown compounds, and then TcVP1 decolorized these colored intermediates to colorless. Following extended reactions, the absorbance corresponding to the conjugated double bond from phenyl (250-300 nm) decreased, indicating that aromatic rings were also degraded. These findings provide important new insights into microbial decolorizing mechanisms and may facilitate the future development of treatment strategies for dye wastewater.

The reactants produced by action of a purified unique dye-decolorizing peroxidase, DyP, on a commercial anthraquinone dye, Reactive Blue 5, were investigated using electrospray ionization mass spectrometry (ESI-MS), thin-layer chromatography (TLC), and (1)H- and (13)C- nuclear magnetic resonance (NMR). The results of ESI-MS analysis showed that phthalic acid, a Product 2 (molecular weight 472.5), and a Product 3 (molecular weight 301.5), were produced. Product 2 and Product 3 were generated by usual peroxidase reaction, whereas phthalic acid was generated by hydrolase- or oxygenase-catalyzed reaction. One potential associated product, o-aminobenzene sulfonic acid, was found to be converted to 2,2'-disulfonyl azobenzene by ESI-MS and NMR analyses. From these results, we propose, for the first time, the degradation pathway of an anthraquinone dye by the enzyme DyP.

Sequential observation of spontaneous endometrial adenocarcinoma development revealed a clear, hormone-dependent, histogenetic pathway in Donryu rats. The first histological changes of the uterine endometrium appeared in both the lining epithelium and uterine gland of the endometrium at 6 mo of age, along with the beginning of persistent estrus. These changes included areas of tall columnar epithelium and gland formation in the lining epithelium as well as metaplastic change in the uterine gland. At 8 mo of age, endometrial hyperplasias were found, with subsequent increase in both incidence and degree. At 8-10 mo of age, hyperplasias were all within the limit of grade + +. After 12 mo of age, however, severe hyperplasias (grade + + +) began to increase markedly, and adenocarcinomas developed at 15 mo of age. The findings thus suggest that uterine endometrial adenocarcinomas arise from hyperplastic lesions, which should therefore be regarded as preneoplastic, as in the human case. Sequential analysis of plasma gonad steroids also ascertained a link between the appearance of these lesions and an increased estrogen : progesterone ratio, suggesting that estrogen may play an important role in development of both hyperplastic and neoplastic lesions. In Fischer-344 rats used for comparative assessment of strain differences, neither advanced histological changes nor hormonal changes were evident.

In addition to spontaneous uterine endometrial adenocarcinomas at a high incidence (35.1%), development of endometrial hyperplasia/adenoma was also frequently detected in rats of the Donryu strain. The total yield of all observed proliferative endometrial lesions was very high (60.6%). The tumors arose commonly in the uterine horn of aged rats. Histologically, most demonstrated glandular structures, consisting of cuboidal or columnar cells with weak eosinophilic or basophilic cytoplasm and large nuclei. In about half of the animals with adenocarcinomas, metastasis to remote organs such as the lung was observed. Histological examination of the ovary and vaginal epithelium revealed ovarian cysts, atrophy of the ovary and cornification of the vaginal epithelium more frequently in rats with endometrial carcinomas than in animals without tumors. These findings indicate that adenocarcinoma development in Donryu rats is associated with endocrine imbalance [increased serum estrogen: progesterone (E2:P)ratios]. By comparative investigation of strain differences, it was confirmed that irregular estrous cycles began earlier with higher incidence in Donryu rats than in F344 rats, a low-incidence strain. Histological findings of the ovary and vaginal epithelium also suggested relatively increased estrogen levels in Donryu rats compared to F344 rats. Estimated plasma values of gonad steroids showed that the E2:P ratio in Donryu rats at 12 months of age was about five times that in F344 rats. These results therefore indicate that hormone imbalance, particularly an increased E2:P ratio, may play an important role in the spontaneous occurrence of endometrial adenocarcinoma in Donryu rats.

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