In order to make a toxicological assessment of organic sulfur compounds in heavy oil various conventional biological tests were applied. (1) Killifish test for median tolerance limit (TLm 24hr). (2) Drosophyla growth test. (3) Chicken embryo assay. (4) Mutagenic activity to Salmonella typhimurium TA 98. (5) Sea urchin egg growth test. The results were as follows: TLm 24hr of C class heavy oil corresponded to around 80ppm, while A and B class heavy oil gave over 100ppm. TLm 24hr of dibenzothiophene was 17ppm. No significant effects of heavy oils on the developmental stage of Drosophyla were observed. In the sea urchin egg growth test, the effective concentration of dibenzothiophene was 25ppm, i. e., this compound was more toxic to marine or fresh water organisms than heavy oil itself. Higher boiling distillate oil such as B and C class heavy oil showed higher toxicity to chicken embryos than A class heavy oil. In the tests for mutagenic activity using TA 98, B class heavy oil showed slight mutagenicity at a concentration of 500μg/plate. Sulfur compounds (benzothiophene and dibenzothiophene) and sulfur containing oil fraction from heavy oil appeared to exhibit no mutagenicity with respect to TA 98.
It has been pointed out that patterns of polychlorobiphenyls (PCB) in the blood of “Yusho” (polychlorobiphenyl-poisoning) patients differ from those of normal adults. Gaschromatography using a 5m column packed with 2% Apiezon L showed that the relative concentrations of 3, 4, 2′, 4′, 5′-pentachlorobiphenyl (peak 25) and 3, 4, 2′, 3′, 4′-pentachlorobiphenyl (peak 28) in the blood of Yusho patients were lower than in normal adults; this was considered to be due to coexisting polychlorodibenzofurans (PCDF) in Yusho oil. To clarify this, PCB and PCDF fractions were separated from Kanemi rice oil (Yusho oil) which had caused Yusho. Yusho oil, rice oil with PCB (154ppm) or rice oil with PCB (154ppm) +PCDF (2ppm) were given orally to different groups of mice at a dose of 0.1ml per body per day for 9 days, and PCB residues in the adipose tissue and liver were examined on the 1st, 7th and 35th days after the termination of dosing. The characteristic PCB patterns of Yusho patients were obtained from the groups given Yusho oil and PCB+PCDF, but not from the group given PCB alone. Less chlorinated biphenyls, peak 25 and peak 28, decreased with time during the experiment, but 3, 4, 2′, 3′, 4′, 5′-hexachlorobiphenyl (peak 47) and 2, 4, 5, 2′, 3′, 4′, 5′-heptachlorobiphenyl (peak 49) increased.
In 1977, there was an outbreak of photosensitivity dermatitis by among persons who had ingested a certain brand of chlorella tablets. At least 23 patients were identified in and around the Tokyo metropolitan area. The unconsumed chlorella tablets collected from these patients invariably induced photosensitivity dermatitis in mice. These chlorella tablets contained large amounts of pheophorbide and its ester, which are well-known phototoxic pigments derived from chlorophyll, while chlorella tablets from another maker, which contained less than a tenth as much pheophorbide induced no photosensitivity lesion in mice. Furthermore, a linear dose response relationship was observed between the severity of photosensitivity dermatitis in experimental animals and the content of pheophorbide in chlorella administered. However, the reaction induced by pheophorbide extracted from the suspect chlorella was weaker than that induced by the original chlorella sample. It was confimed that ethanol, used in forming granules of chlorella before pelleting, activates chlorophyllase, which in turn hydrolyzes chlorophyll naturally contained in chlorella into pheophorbide and its ester. We concluded that the photosensitivity dermatitis was caused by the chlorella tablets, which had been treated with ethanol. We believe that pheophorbide and its ester found in large amount in these tablets, have played a major role in inducing the photosensitivity dermatitis.
Both protein and carbohydrate were necessary for sporulation and enterotoxin production of Clostridium perfringens NCTC 8239 in the ligated intestine of the rabbit. The administration of 108 vegetative cells suspended in 2ml of skim milk was confirmed to induce positive reactions consistently. However, skim milk dialysate, which contained the same amount of protein as the 10% skim milk, had no effect. An addition of 10% lactose to the skim milk dialysate restored the effect. Proteose peptone (Difco), peptone for toxin production (Mikuni), meat extract (Wako) and purified casein (Merck) could replace the skim milk dialysate. In addition to lactose, raffinose and fructose were effective as carbon sources whereas galactose, sucrose and glucose were less effective. Enterotoxin was not produced in the ligated intestine when the vegetative cells were administered together with any one of these carbohydrate or protein sources alone.
In September and October 1977, two outbreaks of food poisoning due to Bacillus cereus occurred in Osaka city. Outbreak 1: On 4th of September 1977, 9 out of a total 13 of employees (69.2%) in two factories became ill 0.5 to 3 hours after taking lunch. Vomiting was the main clinical symptom. B. cereus was isolated (1.4×107 per gram) from the suspect lunches. Fecal specimens collected from 2 patients on the 3rd day of illness were positive for B. cereus. No other known food-poisoning organism was found either in the lunch or in the feces of patients. Outbreak 2: On 9th of October 1977, 211 out of 1809 persons (11.7%) who had eaten a packed lunch (‘Onigiri’, ‘Maki-sushi’ and ‘Inari-sushi’) complained of nausea and vomiting after 0.5 to 6 hours. B. cereus were isolated from ‘Onigiri’ (4.7×108/g), ‘Maki-sushi’ (1.3×104/g) and ‘Inari-sushi’ (3.7×104/g), and from 18 out of 21 fecal specimens (85.7%) collected from the patients on the 1st to 3rd day of illness. All isolates from outbreaks 1 and 2 had the same biological characteristics. Culture supernatants of the two isolates showed heat-sensitive lethal activity to mice on intraperitoneal or intravenous injection.
Amaranth, ponceau 3R, ponceau R and light green SF stimulated RNA synthesis in vitro. Experiments were performed to clarify the mechanism of this effect. 1) Amaranth and ponceau 3R stimulated in vitro RNA synthesis at 1mM in rat liver chromatin as well as in isolated rat liver nuclei. Accordingly, the stimulation of RNA synthesis by these dyes is presumed not to depend on change of the nuclear envelope, which may result in an increase in substrate incorporation into nuclei. On the other hand, light green SF did not stimulate RNA synthesis in rat liver chromatin; the mechanism of stimulation of RNA synthesis by this dye may therefore differ from those of ponceau 3R and amaranth. 2) Amaranth, ponceau 3R and sunset yellow FCF had no effect on DNase 1 or nuclear endogenous nuclease activity. Chromatin and isolated nuclei incubated with ponceau 3R or amaranth are about twice as susceptible to DNase I attack as those incubated without dye or with sunset yellow FCF. 3) Amaranth, ponceau 3R and sunset yellow FCF had little effect on the RNase activity of isolated rat liver nuclei. This result suggests that RNase has little or no involvement in the stimulation of RNA synthesis. It is suggested that the stimulating action of these dyes on RNA synthesis in isolated rat liver nuclei increases in proportion to the amount available for transcription.
The determination of bismuth and lead in foods was examined using a flameless atomic absorption spectrometer equipped with a carbon tube. To elminate the matrix effect, which is observed in direct measurements of acid test solutions, the organic layer obtained by extraction of a test solution (pH ca. 8) with a sodium diethyldithiocarbamic acid (DDTC) -methylisobutylketone system was injected into a carbon tube. Examination of ashing and atomizing conditions gave optimum sensitivities of 3 (bismuth) and 5ppb (lead) for 1% absorption. Coefficients of variation obtained by repeated extractions and injections were 1.9 (bismuth) and 3.4% (lead). Linear standard curves were obtained up to 120 (bismuth) and 100ppb (lead). Although the coexistence of chelating metal ions with DDTC caused some interference it is possible to detemine these metals with fairly good accuracy by a calibration method without using standard addition procedures when this method is used for the analysis of bismuth and lead in modified milk powder.