Mitsuhiro Tsuda

Liver fluke infection and cholangiocarcinoma : Model of endogenous nitric oxide and extragastric nitrosation in human carcinogenesis

Cancers arising during bacterial, viral and parasitic infection provide useful models to investigate the link between inflammation and carcinogenesis. Because the inflammatory agent is known, relationships between immune responses, the production of DNA-damaging agents, such as nitric oxide, oxygen radicles and N-nitroso compounds, and cancer risk can be explored. This paper first describes the close relationship between infection with the liver fluke, Opisthorchis viverrini, and cholangiocarcinoma in humans. Data are then presented which demonstrate an elevation in levels of salivary nitrite and urinary and plasma nitrate among men with moderate and heavy liver fluke infections compared to uninfected controls which was absent 4 months after the parasites were cleared with praziquantel. Because of the strict control over subject selection and dietary intake plus the absence of the increase following treatment, we conclude that the higher levels of nitrate and nitrite reflect endogenous generation of nitric oxide resulting from liver fluke infection. Excess nitric oxide generation in the inflamed tissue is likely to lead directly to the formation of N-nitroso compounds mediated by activated macrophages. Further work will attempt to demonstrate a link between this increase and both parasite-specific immune responses and the risk of cancer.

Formation of thiazolidine-4-carboxyiic acid (thioproline), an effective nitrite-trapping agent in human body, in Parkia speciosa seeds and other edible leguminous seeds in Thailand

Abstract The amounts of thioproline, an effective nitrite-trapping agent in the human body, in various edible leguminous seeds have been determined. Thioproline in uncooked Parkia spedosa , the most popular seeds eaten in the south of Thailand, was undetectable but increased markedly to 0.14 ±0.02 mmmol/100 g after boiling. The formation of thioproline was effectively inhibited by the addition of N -ethylmaleimide, a thiol-trapping agent. The uncooked Parkia speciosa seeds contained substantial amounts of formaldehyde and thiol compounds and the amounts decreased after boiling. The contents of thioproline in other uncooked edible leguminous seeds, Parkia javanica and Archidendron clypearia , were 0.006 ± 0.002 and 0.31 ± 0.11 mmol/100 g, respectively and increased to 0.54 ± 0.2 and 2.95 ±0.8 mmol/100 g after boiling. Archidendron jirringa and Leucaena leucocephala , both uncooked and boiled, showed no detectable amount of thioproline.

Increase in the levels of N-nitrosoproline, N-nitrosothioproline and N-nitroso-2-methylthioproline in human urine by cigarette smoking

The effects of cigarette smoking on the urinary excretions of N-nitrosoproline (NPRO) and the sulfur-containing N-nitrosamino acids, N-nitrosothioproline (NTPRO) and N-nitroso-2-methylthioproline (NMTPRO), in a male volunteer were examined. This subject smoked for 20 non-consecutive days and did not smoke for another 20 non-consecutive days during a 4-month-period, and 24-h urine samples were collected. On the days of urine collection, he was given a fixed diet. On the smoking days, the urinary levels of NPRO, NTPRO and NMTPRO increased significantly from 1.1 +/- 0.5 (mean +/- S.D.) micrograms/day to 1.8 +/- 0.9 (P less than 0.01), from 3.9 +/- 1.1 to 8.7 +/- 4.6 micrograms/day (P less than 0.001), and from 5.6 +/- 1.9 to 8.5 +/- 4.1 micrograms/day (P less than 0.01), respectively. Thus, the total amount of these 3 N-nitrosamino acids in the urine also increased significantly by smoking from 10.6 +/- 2.8 to 19.0 +/- 6.5 micrograms/day (P less than 0.001). These results indicated that NOx in cigarette smoke can contribute to in vivo formation of N-nitroso compounds.

Lipopolysaccharide-associated elevation of serum and urinary nitrite/nitrate levels and hematological changes in rats

The objective of this study was to assess the relationship between lipopolysaccharide (LPS)-induced elevation of nitric oxide (NO) levels and hematological changes. Twenty-four h following i.p. treatment of LPS (1 mg/kg body wt.), nitrite/nitrate (NO2-/NO3-) levels in the serum and urine of rats were, respectively, increased to 11 and 50 times those of control. Time-dependent decrease of white blood cells (80% of control), lymphocytes (40% of control), and platelets (35% of control) was also observed, while a significant increase of neutrophils (330% of control) and monocytes (650% of control) occurred during the 24-h post-treatment period. These results suggest that LPS-induced increase of NO2-/NO3- levels and coincident hematological changes may compromise immune functions.

Formation of Thioproline, Effective as a Nitrite-Trapping Agent in the Human Body, in Various Cooked Foods

Thioproline (TPRO, thiazolidine-4-carboxylic acid) and methylthioproline (MTPRO) are the condensation products of cysteine with formaldehyde and acetaldehyde, respectively. TPRO and MTPRO are thought to be “frozen” cys teines because they liberate cysteine in the liver and protect the liver. We identified N-nitrosothioproline (NTPRO) and N-nitrosomethylfhioprolines (cis and trans) as the major N-nitroso compounds (NOC) in human urine. NTPRO is a nonmutagenic NOC, and thus is probably noncarcinogenic, like N-nitrosoproline. Nitrosation of TPRO is nearly 1000 times faster than that of proline. NTPRO is excreted into urine with out further metabolic change, and has been used as a sensitive monitoring probe like N-nitrosoproline for endogenous for mation of NOC. TPRO and MTPRO may play an important role in cancer prevention due to their effective nitrite-trapping ca pacity in the human body. The anticarcinogenic effect of TPRO has experimentally demonstrated by inhibition of stomach cancer induced by coadministration of benzylmethylamine and nitrite in rats. TPRO was ubiquitously found in various cooked foods, such as vegetables (~2ppm), cod (~5ppm), shiitake mushrooms (~10ppm), and liver-based foods (~15ppm). Especially high contents were detected in djenkol beans (~800ppm), which are eaten in Indonesia, and several types of leguminous beans (>3500ppm) eaten in southern Thailand.

pH-dependent degradation of nitrosocimetidine and its mechanisms.

The degradation of nitrosocimetidine (NC) and its mechanism were found to be strongly dependent upon pH by monitoring NC and its degradation products by high‐performance liquid chromatography (HPLC). NC was relatively stable at neutral pH, but it degraded rapidly under both acidic and alkaline conditions. In a strongly acidic solution, the degradation was shown to be entirely by denitrosation, but under alkaline conditions scarcely any denitrosation was observed and various other degradation products were found. At neutral pH, both these degradation mechanisms were observed. In neutral solution, the presence of thiol compounds greatly shortened the half life of NC, and enhanced its denitrosation. The high degradation rate in acidic solution, the strong influence of thiol groups, and the preference of denitrosation at pH 0.3‐5 can explain the discrepancy between the in vitro genotoxicity and the lack of carcinogenicity of NC.