Naoko Harada

Ascorbate radical levels in human sera and rat plasma intoxicated with paraquat and diquat

To clarify the toxicological mechanism of action of paraquat and diquat, the level of ascorbate radical, an oxidized product of ascorbic acid, was examined using the electron spin resenance (ESR) method. The ascorbate radiacal level increased to twice the normal level in sera from humans acutely intoxicated with a 1∶1 mixture of paraquat and diquat, and to more than 1.5 times the normal level in rat plasma containing moderate levels of paraquat. The dosage level for rats was 0.025% paraquat dichloride in the diet. The ascorbate radical in both intoxicated human sera and rat plasma decayed much faster than that in normal samples

Acute-phase reactant proteins and antioxidants in rats intoxicated chronically with paraquat

Paraquat dichloride at 250 ppm in the diet was fed continuously to rats. Though no apparent effect of paraquat was observed until 10 d, some rats then began to show several symptoms such as diarrhea, anorexia, epistaxis, and hypokinesia, and in some cases rats died after this period. The biochemical examination of plasma components revealed appreciable changes in the concentrations of an acute-phase reactant protein and some vitamins that act as antioxidants. alpha-Cysteine proteinase inhibitor increased by 5-fold, and vitamin C and its radical increased by 1.5- and 1.7-fold, respectively, whereas alpha 1 proteinase inhibitor decreased slightly. Paraquat enhanced the cysteine proteinase inhibitor levels in lung, liver, and kidney by 6.2-, 6.0-, and 4.5-fold of control, respectively. Among three components of alpha-cysteine proteinase inhibitor, the T kininogen level of treated rat plasma was about eight-fold higher than control, whereas the high-molecular-weight kininogen level was unchanged. The large increment of T kininogen was also seen in lungs of the treated rats.

Determination of molar absorptivities of radicals of 18 phenothiazine derivatives

Abstract A new colorimetric method is described for the quantification of 18 different phenothiazine derivatives. The phenothiazine derivatives changed immediately to various different colored radicals following their oxidation with 2,3-dichloro-5,6-dicyano- p -benzoquinone in 47% or 66% perchloric acid solution. The radicals produced were very stable. The extent of color fading after 1 h was less than 2% for all 18 radicals, and that after 24 h was less than 2% for 16 radicals. The absorption maxima of the 18 radicals in the visible region showed a linear concentration dependence in the range 0.5–10 μg/ml phenothiazine. The molar absorptivities of these radicals were determined in the ultraviolet and visible regions. With the exception of two derivatives studied, these radicals exhibited one prominent peak in the wavelength range 280–300 nm having a molar absorptivity ∼40 000 while the other peak occured between 500–640 nm having a molar absorptivity ∼10 000. These 18 phenothiazine derivatives were classified into seven groups according to the wavelength of the absorption maximum in the visible region.

Effect of dietary paraquat on a rat mutant unable to synthesize ascorbic acid

To obtain some insight into the toxicity of paraquat (PQ) in humans, PQ dichloride at 250 ppm in the diet was administered to both normal (NO) rats and ODS-od/od (OD) rats which are unable to synthesize ascorbic acid (AsA). Firstly, OD rats and NO rats treated with PQ were compared with untreated NO rats (CO). Only OD rats displayed several symptoms of PQ poisoning such as anorexia, hypokinesia, diarrhea, epistaxis, tremor and their pili became rough about 9 days after. Their cysteine proteinase inhibitor level in plasma and lung increased to 2- and 6-fold, respectively, of CO. In contrast, NO rats treated with PQ resembled CO rats, and their cysteine proteinase inhibitor levels were unchanged until 11 days. After this period they began to display symptoms. Secondly, OD rats fed with different amounts of AsA were compared. Excess AsA delayed the onset of symptoms by only 1 day. Thirdly, the day of onset of symptoms was found to be influenced with the weight of rats.

A new diquat derivative appropriate for colourimetric measurements of biological materials in the presence of paraquat

Abstract A new colourimetric method is described for the quantification of diquat using a yellow-coloured derivative produced by heating diquat in alkaline solution at 80°C. The absorption maximum of the yellow derivative is 420 nm and the molar absorption coefficient is 2.76 × 104 (0.15 in 1 μg diquat/ml with 1 cm light path). The absorption at 420 nm shows a linear concentration dependence in the range 0.1–10 μg/ml and fading of the colour is about 5% after 1 h. Under the same conditions, paraquat does not produce any coloured products. The concentration of diquat in the solution containing both diquat and paraquat can be determined by the absorption of diquat derivative at 420 nm without interference from paraquat. By adding sodium dithionite to the solution the concentration of paraquat can be determined by the absorption of paraquat radicals at 600 nm without interference from diquat, because the yellow derivative does not react with dithionite. This yellow diquat derivative can be extracted completely with cyclohexanol by saturating the solution with Na2SO4. The absorption maximum in cyclohexanol shifts to 440 nm with the same molar absorbance and the same half-band width as in water. Fading of the colour is less than 5% after 24 h in cyclohexanol. Perchloric acid (3%) and trichloroacetic acid (4.5%) which are often used for deproteinization of tissue homogenates, do not inhibit production of the coloured derivative at pH 13.5 or extraction of the derivative with cyclohexanol. This method is suitable for a quick determination of small amounts of diquat in tissues, since the extraction with cyclohexanol not only concentrates the derivative rapidly but also quite efficiently eliminates the coloured substances in tissue homogenates. The detection limit of diquat is 0.02 μg/ml for blood and 0.05 μg/g for liver when 1 ml or 1 g is used for analysis. In three human cases of fatal intoxication, both paraquat and diquat were quantified using 50 μl of serum. In non-toxic dosing of diquat to rats for 14 days, the diquat level was highest in the spleen followed by the kidneys.

Dietary Mg and/or K restriction enhances paraquat toxicity in rats

Abstract Effect of mineral restriction was studied to clarify which mineral in the diet is most indispensable in preventing paraquat (PQ) toxicosis. ODS rats were chosen as the experimental animal owing to the inability to synthesize vitamin C similarly to humans. Rats were fed with either mineral-adequate or restricted diets dosed with 125 ppm PQ. The mineral-adequate diet was based on the American Institute of Nutrition-76, and the restricted diet was one-half the amounts. Measurements were made on the onset day of PQ toxicosis, body weight changes during the feeding experiment, and changes of two acute phase reactant proteins – cysteine proteinase inhibitor and α1-proteinase inhibitor. The minerals tested were divided into three classes: I, largely needed, Ca, K, Na, and Mg; II, moderately needed, Mn, Fe, Zn, and Cu; and III, minutely needed, Cr and Se, respectively. Rats fed with a Mg-restricted diet showed a severe toxicosis but those with a K-restricted diet, a mild toxicosis. No appreciable effect was observed by restriction of other minerals. A synergistic effect was observed in the restriction of Mg and K.

Diquat increases cysteine proteinase inhibitors greatly in rat plasma and tissues

Biochemical and gross pathological effects of diquat were studied with special attention to cysteine proteinase inhibitor level which was often increased in acute and chronic disorder. Diquat was fed continuously to rats at the dose of 1000 ppm in the diet. After 10 days, anorexia and severe diarrhea were observed but epistaxis and hypokinesia were not apparent. The rats were killed after feeding the diet for 13.5 days and plasma components such as acute phase reactant proteins and some vitamins which act as antioxidants were examined. The results showed that α-cysteine proteinase inhibitor (α-CPI) increased to 9-fold and vitamin C radical increased to 1.6-fold, whereas α1 proteinase inhibitor (α1-PI) decreased to 0.9-fold and vitamins C and E were the same as the control. Among three components of α-CPI, the T kininogen level in intoxicated rat plasma was about 20-fold, whereas the high molecular weight kininogen level was about 2-fold of the control. Diquat also enhanced the cysteine proteinase inhibitor (CPI) level to 20-fold in kidney and to 7- to 10-fold in the other organs. The large increment of T kininogen in these organs was also confirmed immunologically. The kidney showed a granular degeneration and its weight increased to 1.2-fold of control. The other organs showed neither gross pathological alteration nor weight change, compared with the control. The diquat distribution was highest in spleen and next highest in kidney among several organs. These results were compared with those caused by paraquat.

Effect of Dietary Paraquat on a Rat Mutant Unable to Synthesize Vitamin C

The toxicity of paraquat (PQ) was examined using ODS-od/od (OD) rats unable to synthesize vitamin C (VC). First, OD rats and normal rats treated with 250 ppm PQ in the diet were compared. The onset of toxicosis of OD rats was 6 days earlier than that of normal rats. Second, the degree of toxicity was compared by administering PQ 35, 125, or 250 ppm to OD rats receiving VC 300 ppm. Only the rats dosed with PQ 250 ppm exhibited toxicosis. The cysteine proteinase inhibitor levels of intoxicated rats increased, whereas the VC levels decreased in plasma, liver, and especially lung, respectively. Third, the degree of toxicity of PQ 250 ppm to OD rats was compared by changing the supplementation of VC as 150, 300, or 3000 ppm. The excess amount of VC delayed the onset of toxicosis only 1 day. The VC levels of the VC-oversupplied group in plasma and liver were fourfold and twofold of control, respectively; but the level was only 0.6-fold of control in lung, which is the target organ of PQ.