Evaluation of the effect of steviol on chromosomal damage using micronucleus test in three laboratory animal species.

The chromosomal damage activity of steviol, a product of enzymatic alteration of stevioside, a natural non-caloric sweetener was reevaluated by using a bone marrow micronucleus test in both male and female hamsters, rats and mice. The micronucleus test is used widely as a rapid and efficient alternative in chromosome analysis for detecting in vivo cytogenetic damage. Steviol at the dose of 4 g/kg body weight for hamsters and 8 g/kg body weight for rats and mice showed no effect on the frequencies of micronucleus formation in bone marrow erythrocytes of both male and female hamsters, rats and mice. Moreover, there was also no apparent change in the PCEs:NCEs (polychromatic erythrocytes:normochromatic erythrocytes) ratio of the male animals of all three treated species at 24, 30, 48 and 72 hour intervals. However, steviol at the given dose can cause significant reduction of PCEs to NCEs ratio of the female hamsters at 72 hours and female rats and mice at 48 and 72 hours after receiving steviol orally. From these results, it could be proposed that steviol at the given dose to the treated animals produced adverse metabolites and these metabolites could reach the bone marrow, the target organ for micronucleus test. These metabolites also exhibited a slightly cytotoxic effect but not clastogenic effect to the bone marrow erythrocytes.

Developmental toxicity of steviol, a metabolite of stevioside, in the hamster.

The developmental toxicity of steviol, a metabolite of stevioside, was studied in hamsters. Pregnant hamsters were intubated with steviol at dose levels of 0, 0.25, 0.5, 0.75 and 1.0 g/kg BW/day on days 6-10 of gestation. Steviol at doses of 0.75 and 1.0 g/kg BW/day were highly toxic to both dams and fetuses. Significant decrease of maternal body-weight gain during the experimental period (days 6-14) and high percentage of maternal mortality indicated the general toxicity of these two high doses. The number of live fetuses per litter and mean fetal weight also significantly decreased in the steviol-treated animals at doses of 0.75 and 1.0 g/kg BW day. The animals treated with an intermediate dose (0.50 g/kg BW/day) exhibited less signs of maternal and developmental toxicity than the two high doses (0.75 and 1.0 g/kg BW/day). One craniomeningocele was found in a fetus under the maternal toxic condition in steviol-treated at a dose of 0.75 g/kg BW/day. Neither the skeleton nor visceral development of the offspring was affected by steviol treatment except delayed ossification of the xiphoid (bifid) and long bones of the limbs and supernumerary thoracic ribs (14th ribs) tended to be increased at doses of 0.5 to 1.0 g/kg BW/day steviol. No dose-related teratogenesis was detected. From the result of the present study concerning maternal toxic condition and embryotoxicity, an oral dose of 0.25 g steviol/kg BW/day is regarded as having no observable effect. This steviol-treated dose is derived from stevioside 625 mg/kg BW/day which is approximately 80 times higher than the suggested acceptable daily intake of stevioside for humans (7.938 mg/kg BW/day).

Lack of mutagenicity of stevioside and steviol in Salmonella typhimurium TA 98 and TA 100.

Stevioside, a sweet-tasting diterpene glycoside derived from Stevia rebaudiana, and steviol, a product from enzymatic hydrolysis of stevioside, were tested for mutagenic activity by the in vitro Ames test, a preincubation method, using Salmonella typhimurium TA 98 and TA 100 as the tester strains, either in the presence or absence of metabolic activating system derived from the sodium phenobarbital and 5,6-benzoflavone pretreated liver S9 fractions from various animal species including rat, mouse, hamster and guinea pig. Stevioside and steviol at the concentrations up to 50 mg and 2 mg per plate, respectively showed no mutagenic effect on both tester strains either in the presence or absence of metabolic activating system. However, at the high concentration both stevioside and steviol showed some toxic effects on both tester strains. The toxic effect was decreased in the presence of the metabolic activating system.

Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species.

The acute toxicity of stevioside and steviol (a product of enzymatic hydrolysis of stevioside) was investigated in three animal species including rat, mouse and hamster. The susceptibility to stevioside and steviol acute toxicity in both sexes of these animal species was compared. The animals were treated intragastrically with stevioside or steviol and general signs and symptoms were observed. The numbers of dead animals were recorded within a period of 14 days after administration for estimation of LD50. Stevioside at a dose as high as 15 g/kg BW was not lethal to either mice, rats or hamsters. Hamsters were found to be more susceptible to steviol than rats or mice. LD50 values of steviol in hamsters were 5.20 and 6.10 g/kg BW for males and females, respectively. In rats and mice, LD50 values of steviol were higher than 15 g/kg BW in both sexes. Histopathological examination in the kidney of hamsters induced by steviol revealed severe degeneration of the proximal tubular cells. These structural alterations were correlated with the increases in serum blood urea nitrogen (BUN) and creatinine. Therefore, the possible cause of death induced by steviol might be due to acute renal failure.

Mutagenic activity of newly synthesized sulfa drugs to Salmonella typhimurium.

The mutagenic activity of seven newly synthesized sulfa drugs was studied in Salmonella typhimurium, using forward mutation to 8-azaguanine (8-AG) resistance and reversion mutation assays (Ames test) both in the absence and presence of Aroclor induced rat liver S9. In forward mutation assays, N1-methylsulfanilamide, N4-acetyl-N1-methylsulfanilamide and N4-acetyl-N1-diethylsulfanilamide were mutagenic to S. typhimurium TM677 both in the presence and absence of metabolic activation while N4-acetylsulfanilamide, N1-diethylsulfanilamide and 4-nitro-N-2-pyridinylbenzenesulfonamide [2-(p-nitrobenzenesulfonamido)pyridine] were mutagenic only in the presence of metabolic activation. But 2-(N4-acetylsulfanilamido)pyridine was mutagenic in neither the presence nor the absence of metabolic activation. However, none of the seven compounds had any mutagenic effect on S. typhimurium TA98 or TA100 in the absence or presence of metabolic activation, by the Ames test preincubation method. The relationship between the structure of the compounds and their mutagenic activity is also discussed.

Comparative horizontal starch gel isoenzyme electrophoresis of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Foot muscle tissue extracts from six lymnaeid species of the Indo-Pacific region [Lymnaea (Bullastra) cumingiana and L. (Radix) quadrasi from the Philippines, L. (R.) rubiginosa from Indonesia and Thailand, and L. (R.) viridis from Guam and Hong Kong] were subjected to horizontal starch gel isoenzyme electrophoresis and assayed for seven isoenzymes (AcP, AlP, CA, EST, LAP, CAT and GOT) to elucidate their taxonomic relationships. L. cumingiana exhibited banding patterns for EST, LAP and CAT uniquely different from the rest, thus supporting the hypothesis that it is a distinct species. Zymogram patterns for AlP, CA, EST and LAP attest to the close affinity between L. quadrasi and L. rubiginosa (Indonesia and Thailand). Minor differences suggest a closer relationship between the two geographical strains of L. rubiginosa than with L. quadrasi, lending support to the hypothesis that L. quadrasi is inseparable as a race or variety from the typical L. swinhoei Adams, which in turn is but a race of L. auricularia, which also encompasses L. rubiginosa. The two geographical strains of L. viridis from Guam and Hong Kong showed the greatest consistency with regards to similarity and congruence in banding patterns. Non-specific esterases (EST) were the most useful in distinguishing the six species from each other.

Comparative radular morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

The radular morphology of Lymnaea (Bullastra) cumingiana was compared to that of five other Indo-Pacific lymnaeid "species", namely: L. (Radix) quadrasi (Philippines), L. (R.) rubiginosa (Indonesia and Thailand) and L. (R.) viridix (Guam and Hong Kong) in order to investigate the taxonomic relationship among the six species. Although all six species uniformly exhibited a unicuspid, slightly asymmetrical central (rachidian) tooth and tricuspid laterals, interesting differences were noted among the outer marginals. These were observed to be uniquely bicuspid in L. cumingiana, predominantly tricuspid in L. quadrasi, tetracuspid in L. rubiginosa (Indonesia and Thailand) and multicuspid in L. viridis (Guam and Hong Kong). Thus, the results support the hypotheses that L. cumingiana is a unique species compared to the rest, that L. quadrasi is closely related to L. rubiginosa (Indonesia and Thailand) and that the two geographical isolates of L. viridis have not diverged. Radular morphology was therefore found to have a limited significance in elucidating the taxonomic relationship between the six groups of lymnaeids studied.

Comparative shell morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Comparative shell morphology using both quantitative and qualitative parameters was employed to investigate the taxonomic relationship between the endemic Philippine species, Lymnaea (Bullastra) cumingiana and five other lymnaeid "species" in the Indo-Pacific region, namely: L. (Radix) quadrasi (Philippines). L. (Radix) rubiginosa (Indonesia), L. (Radix) rubiginosa (Thailand), L. (Radix) viridis (Guam) and L. (Radix) viridis (Hong Kong). Fifty randomly chosen adult specimens of each species were studied and compared, although only field-collected specimens were studied for the first four groups and laboratory-raised specimens for the last two group. Results strongly suggested that L. cumingiana is a distinct species among the rest. L. quadrasi, L. rubiginosa (Indonesia) and L. rubiginosa (Thailand) exhibited great affinity towards each other. Likewise, the two geographical isolates of L. viridis were practically identical to each other except for some minor size differences.

Ecological observations on Lymnaea (Bullastra) cumingiana.

Field surveys conducted at Echague, Isabela and San Pablo, Laguna revealed that Lymnaea (Bullastra) cumingiana, the natural second snail intermediate host of Echinostoma malayanum in the Philippines, exhibits a moderate degree of diversity in its choice of habitats. Rice fields of all stages of development, stagnant shallow streams and springs are the main areas where the snail can be collected from at Echague, Isabela. However, they were absent in rice fields that had been extensively sprayed with molluscicides to control the "golden apple snail" (Ampullarius canaliculatus). In contrast, they were also very abundant in the highly eutrophic waters of Sampaloc lake, San Pablo, Laguna. L. cumingiana co-exists with various species of insects, snails, fish and plants in these habitats. Information on ecological characteristics affecting its distribution will be useful for those who wish to collect and study this species in the future.

Malacological survey in the Sirikit reservoir, the largest earthfilled dam in Thailand.

Ten species of freshwater molluscs were found in a malacological survey in the Sirikit reservoir in 1985. Among these species, Tricula aperta or Neotricula aperta, intermediate host of human schistosomes, Bithynia (Digoniostoma) siamensis goniomphalos and B. (D.) funiculata, the first intermediate host of Opisthorchis viverrini were not found. It is revealed that most of the habitats in the Sirikit reservoir are not suitable for the survival and colonization of molluscs. Thus few species of edible molluscs in small numbers were found, except for Limnoperna siamensis, which were found in large numbers in the reservoir. Although it is not a medically important species, their attaching in large colonies may reduce the volume of water flowing into the power tunnels and obstruct small pipe lines in the dam area. So it is recommended to further study the life cycle of L. siamensis and to determine suitable molluscicides or biological agents to be used in controlling them.

Malacological survey in the Bhumibol Reservoir, the first dam constructed in Thailand.

Twenty-five species of molluscs were found in a malacological survey in the Bhumibol Reservoir and in the Ping River in front of the Bhumibol Dam and its tributaries in April and August 1985. Among these species, Tricula aperta or Neotricula aperta, intermediate host of human schistosomes and Bithynia (Digoniostoma) siamensis goniomphalos and B. (D.) funiculata, the first intermediate host of human liver flukes, were not found. Although some of the intermediate hosts of mammalian schistosomes, Indoplanorbis exustus and Lymnaea (Radix) auricularia rubiginosa were found, none of them were positive for the parasitic infection. However, the malacological survey in the Bhumibol Reservoir revealed the existence of colonies of edible snails, Filopaludina (Filopaludina) doliaris, F. (F.) filosa and F. (Siamopaludina) martensi martensi along the shoreline of the reservoir. Thus, it is recommended that the local villagers collect them for household consumption or for sale. This practice would augment the food supply and also increase the income of the local villagers in the Bhumibol Reservoir area.

Induction of sister-chromatid exchange in human blood lymphocytes by aqueous extract of palmyrah (Borassus flabellifer) flour.

pPalmyrah palm (Borassus flabellifer) is widely consumed by people in certain tropical countries. The incidence of human malignant lymphomas, mutagenicity and toxicity in rats and bacteria encouraged us to study the potency of palmyrah crude aqueous extracts in inducing sister-chromatid exchanges (SCEs) in human blood lymphocytes in vitro. The extracts induced SCEs in a dose-related manner in both females and males. These effects apparently showed no consistency between batches. This result may be due to the intrinsic variation of different donors in their response to the induction of SCEs by palmyrah extracts. SCE frequency was proportional to chromosome length and SCEs at the centromeric region showed no difficulty in being scored. Concerning methods of short-term cytogenetic testing for detecting mutagenic and carcinogenic chemicals, we found that the SCE test was not more sensitive than the classic chromosome-breakage test.

Intestinal parasitoses among the workers and their families at three electricity generating dams in Thailand.

During September 17, 1980 - May 29, 1981 a total of 986 single stool specimens were collected from workers and their families at three electricity generating dams under the administration of the Electricity Generating Authority of Thailand (EGAT). Parasitological surveys were carried out at the dam sites, namely the Khoa Laem Project, the Srinakarin Dam and the Ubolratana Dam. The overall prevalence at all three sites are moderately high (62.50%, 45.33% and 54.05% respectively). The Khoa Laem project had the highest prevalence of E. histolytica infection (16.16%) which may be the result of poorer sanitary standards for workers of the project under construction. The occurrence of opisthorchiasis at the first two sites situated in central Thailand (12.19% and 7.01%) is the result of influx of migrant northeastern workers. The prevalence of other parasites are not remarkable. However, because of the proximity of the people living close to the large bodies of fresh water, urgent measures should be taken to eradicate the parasitic infections among them in order to prevent the spread through the water.

Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium.

Toxic and mutagenic activities of formaldehyde were studied in Salmonella typhimurium strain TM677, using forward mutation to 8-azaguanine (8-AG) resistance both in the absence and in the presence of Aroclor-induced rat-liver postmitochondrial supernatant (PMS). The results showed that formaldehyde was toxic and mutagenic to the bacteria in both systems, but toxicity and mutagenicity were reduced in the presence of PMS. The minimum concentration required to induce toxicity and mutagenicity was 0.17 mM in the absence of PMS and 0.33 mM in the presence of PMS.

Freshwater snails survey in and around the Cambodian refugee camp, Sakaeo District, Prachinburi Province, Thailand.

The freshwater snails surveys were carried out during September 15, 1981 to December 15, 1981, especially for the intermediate or possible intermediate host of Schistosoma mekongi in and around the Cambodian refugee camp at Ban Kaeng Holding Centre, Sakaeo District, Prachinburi Province. A total of 30 localities were visited and 15 species of freshwater snails were collected. The snails did not shed any cercaria of S. mekongi and none of these was the intermediate host of S. mekongi.

A survey on intestinal parasitic infections in Laotian refugees at Ubon Province, Northeastern Thailand, with special reference to schistosomiasis.

454 stool specimens were collected from the Laotian refugees residing in the refugee camp, Ubon Province, from August 8 to 11, 1978, and were examined by formalin ether sedimentation technique. One of them (0.22%) was infected with Schistosoma mekongi. Since there are freshwater hydrobiid snails, Lithoglyphopsis aperta, the intermediate host of S. mekongi in that area, it is probable that the disease may become established in Northeastern Thailand in the future, which will eventually lead to health and economic problems in Thailand.