2. Biochemistry Division

A variety of environmental as well as endogenous factors are involved in human carcinogenesis. The main purpose of the Biochemistry Division is to assess the role of these factors in human cancer development. The results obtained from these studies are anticipated to lead to the discovery of new ways to prevent human cancer.

Environmental Mutagens

Water from the Katsura River and the Nishitakase River, especially at sites close to a sewage plant, was strongly mutagenic to Salmonella typhimurium TA98 with S9 mix. One and a half kilograms of blue cotton were suspended in the Nishitakase River for 24 hours. The adsorbed materials were then eluted from the blue cotton and the mutagens were successfully isolated with a Sephadex LH-20 column and HPLC with ODS columns. Finally, approximately 60 ug each of two mutagens, compounds I and II, were isolated, and their UV absorption maxima were found to be 390 nm. One ug each of compounds I and II induced 28,000 and 36,000 revertants of TA98 with S9 mix, respectively, and these mutagenic potencies were comparable to those of Trp-P-2 and Glu-P-1. Their structures are now being analyzed. To study the role of carcinogenic heterocyclic amines in human cancer development, MeIQx-DNA adducts in 38 DNA samples obtained from surgical and autopsy specimens were analyzed by the ³²P-postlabeling method. Two surgical specimens, colon and rectum and an autopsy specimen, the kidney, were found to contain 5'-pdG-C8-MeIQx at levels of 14, 18 and 1.8/10¹⁰ nucleotides, respectively.⁽²⁸⁾ This adduct formation may be involved in human carcinogenesis. Methylglyoxal is a direct-acting mutagen in S. typhimurium TA100 and its mutagenicity is markedly enhanced by hydrogen peroxide. ³²P-Postlabeling analysis demonstrated that the guanine residues in DNA were acetylated by methylglyoxal in the presence of hydrogen peroxide in vitro.⁽²⁹⁾
Reports related to the above work can be found in the attached list of references.^(30-35)

Comutagenic Action of Norharman

Norharman, widely distributed in our environment, is mutagenic to neither the TA98 nor the TA100 strain of S. typhimurium, but becomes mutagenic to TA98 with the addition of aniline or its metabolite, phenylhy-droxylamine (PHA), in the presence of S9 mix. DNA adduct formation by norharman with aromatic amines correlates with the comutagenic action of norharman in TA98.⁽³⁶⁾ To elucidate the mechanisms of this comutagenic action, the products resulting from a reaction between norharman and PHA in the presence of S9 mix were separated by HPLC. A product requiring S9 mix in order to exert its mutagenic effect was isolated as a single peak. A mass spectrum of the isolated compound indicated its molecular weight to be 259. A ¹H-NMR spectrum suggested that the compound consisted of a norharman and an aniline molecule, with condensation taking place at N-9 of norharman and C-4 of aniline. The suggested compound was synthesized, and its structure was concluded to be 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole.

Cytotoxic Factor Present in Cabbage Butterfly

Specific bioactive materials are involved in the defense systems of insects. In order to screen for novel cytotoxic substances present in insects, the cytotoxic activity of extracts taken from the pupae and adults of various kinds of butterflies and moths was tested in vitro against human gastric carcinoma TMK-1 cells. Among the species examined, cytotoxicity was limited to Pieris rapae (known as the cabbage butterfly), Pieris napi and Pieris brassicae. When the cytotoxicity of the three developmental stages, larvae, pupae and adults, of Pieris rapae was compared, the pupae showed the strongest activity, with IC₅₀ against TMK-1 cells being at a dilution of 1/10⁶. The active component of the pupae of Pieris rapae was found to be heat-labile and not extractable with organic solvents, but was precipitated with ammonium sulfate and was digested by proteases, suggesting that it is a protein. This cytotoxic factor was named pierisin.⁽³⁷⁾

Poly(ADP-ribose) Polymerase and Cell-cycle Check-point Control

DNA strand breaks immediately activate poly(ADP-ribose) polymerase (PARP). A PARP inhibitor, 3-amino-benzamide, suppresses G1 arrest and enhances G2 arrest after g-irradiation. In the presence of damaged DNA, purified wild-type p53 protein was efficiently poly(ADP-ribosyl)ated. Furthermore, the pRB phosphorylating activity of cdks was down-regulated by purified PARP under DNA damaging conditions. These studies suggest the involvement of PARP in DNA damage signal-transduction. To elucidate the physiological functions of PARP, including the control of cell-cycle check-points, a gene-disruption study of PARP is now in progress using a murine model.

List of papers from this division

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