Comparative cytotoxicity of aflatoxin B1 and saxitoxin in cell cultures.

Human and animal cell cultures were evaluated for their susceptibility to two environmental toxins found as contaminants in human food supplies: aflatoxin B1, a hepatotoxin produced by the mold Aspergillus flavus, and saxitoxin, a paralytic neurotoxin produced by the marine dinoflagellate Gonyaulax catenella. Both toxins cause food poisoning in humans and other animals. The acute cytotoxicity of both toxins was measured and compared by inhibition of cell growth and by progressive cytopathogenicity resulting in cell destruction. Aflatoxin B1 was cytotoxic to all of the 11 primary kidney cultures derived from susceptible animals. The cell growth inhibition 10% values (TD10) ranged from 0.02 to 6.0 micrograms/ml: mouse (TD10 = 0.02 micrograms), guinea pig (0.03 micrograms), rat (0.07 micrograms), hamster (0.16 micrograms), monkey (0.1 microgram), human (0.7-1.5 micrograms), chick (0.05 micrograms), and duck (6.0 micrograms). The corresponding TD50 levels were about 10 times higher concentrations and caused cell destruction within 2 d. Saxitoxin did not induce cytotoxicity manifestations in cultures derived from susceptible species--mouse kidney, human carcinoma HeLa line, chick embryo, and goldfish fin (CAR) cell line--at high concentration levels up to 5 micrograms/ml. When the same toxin preparation at only 1 microgram was injected into mice, the animals died immediately. The results indicate that animal cell cultures are useful for studies of general cytotoxins that affect common essential metabolism but cannot be used to detect environmental toxins that cause toxic manifestations by an interference with specific physiological functions of organ systems.

DDT and immunological responses. 3. Reduced anaphylaxis and mast cell population in rats fed DDT.

Previous work showed that anaphylaxis, mast cell population, and tissue histamine content are reduced in guinea pigs given DDT injections. This study was intended to determine whether dietary intake of DDT causes similar effects. Rats immunized with diphtheria toxoid and fed diets containing DDT at 20 and 200 ppm levels for 31 days did not show effects on their serum antitoxin titers, but the numbers of metachromatically stained, histamine-containing mast cells in mesenteries were reduced: in the 20 ppm group by 46% and in the 200 ppm group by 61%. The severity of anaphylactic shock was also reduced in proportion to the DDT dietary levels, and, thus, the magnitude of mast cells. Apparently, daily dietary DDT intake above 2.2 mg DDT/kg of body weight alters the physiology of mast cells in the rat, and thus affects histamine-mediated reactions.

Protective action of 2,2-bis(parachlorophenyl). 1,1,1-trichloroethane against intoxication of cells by staphylococcal enterotoxin B.

Chlorinated hydrocarbon 2,2-bis(parachlorophenyl)-1,1,1-trichloroethane (DDT) and its derivative 2,2-bis(parachlorophenyl)-1,1-dichloroethane (o,p'-DDD) protected the cells of human embryonic intestine, Henle strain, against the cytotoxic effects induced by staphylococcal enterotoxin B (SEB). The hydrocarbons were incorporated in the culture medium, and the cytotoxicity of SEB was measured by cell destruction and by inhibition of cell growth during incubation for 48 hr. When DDT at nontoxic levels (4 to 25 mug/ml) was added to cultures, 24 or 4 hr prior to or 7 hr after the administration of toxin (100 mug/ml), the cells did not show the characteristic cytotoxicity. A therapeutic effect was also observed with o,p'-DDD added to cultures 9 hr after SEB. Because the cells treated with DDT for a period of 24 hr remained resistant to the toxin when DDT was removed from the cultures, it appears that DDT acts upon the cells rather then upon enterotoxin B. The protective effect of DDT was also observed against the toxin-induced skin erythemas and necrosis in guinea pigs treated with DDT.

Interference by trypsin in the interaction of staphylococcal enterotoxin B and cell cultures of human embryonic intestine.

The inhibitory effect of trypsin on the cytotoxicity of staphylococcal enterotoxin B acting with human embryonic intestine cell cultures was examined. Trypsin treatment of the cells rendered them resistant to enterotoxin for a period of 48 hr. The resistance increased proportionally with increased time of exposure of the cells to trypsin. Neither ethylenediaminetetraacetic acid nor scraping, which were used as alternate means of cell suspension, caused any resistance to the toxin. The effect is enzymatic and appears to be similar to the inhibitory action of trypsin and chymotrypsin on the attachment of polioviruses and coxsackieviruses to HeLa cells.

Interaction of diphtheria toxin with cell cultures from susceptibile and resistant animals.

The cytotoxicity (TD) level of diphtheria toxin for human Chang liver strain was 0.1 guinea pig MLD per ml; for mouse liver NCTC 1469 strain, the TD was 500 MLD per ml. The results of cell culture toxicity correlated well with relative susceptibility of both man and mouse. The initial effects of toxin on the susceptible Chang liver cells were studied at one half the TD level (0.05 MLD per ml). At this low concentration of toxin, the number of cells per culture was reduced slightly below the "0" hr values, whereas the amounts of cell protein, RNA, and DNA were increased. Analysis of the toxin-treated cells indicated an enlargement of the cells. The individual cells contained significantly more protein, RNA, and DNA than the control cells and the cell volume was increased 1.9 times. When purified diphtheria toxin was incubated with the susceptible Chang liver cells and then tested for its biological activity, the results showed an increased diffusion rate in parabiotic culture chambers and definite cytotoxicity to the normally resistant mouse liver cells. The cytotoxicity was neutralizable with antitoxin. The results suggest that the toxin-susceptible cells transform the toxin molecule to a more active derivative which affects the highly resistant mouse liver cell.

Interaction of staphylococcal enterotoxin B with cell cultures of human embryonic intestine.

Schaeffer, W. I. (Massachusetts Institute of Technology, Cambridge), J. Gabliks, and R. Calitis. Interaction of staphylococcal enterotoxin B with cell cultures of human embryonic intestine. J. Bacteriol. 91:21-26. 1966.-The cytotoxic effect of staphylococcal enterotoxin B upon human embryonic intestine cell cultures is characterized by retraction of cells from the monolayer. This is followed by clumping of the retracted cells to form clear areas in the monolayer and finally by sloughing of the clumps from the glass surface. The 50% effective dose of the toxin, determined by protein analysis of the cultures used in titration studies, was found to be between 40 and 60 mug/ml. The cytotoxic property of the enterotoxin was completely neutralized by 3.9 x 10(-5) ml of specific antitoxin per mug of toxin. The cytotoxicity was found to be slightly enhanced by 2.2 g of bicarbonate per liter of Eagle's basal medium (Earle's salt solution level), the absence of serum, the absence of penicillin and streptomycin, and the presence of 2.8 mmoles of calcium in the medium. The cytotoxicity was profoundly influenced by the age of the culture. No cytotoxicity was evident until after 2 days of growth had taken place, when the cell number was approximately 4.0 x 10(5) cells per culture.

The role of methionine deficiency in poliovirus replication in tissue cultures.

The role of methionine in poliovirus infection in HeLa and monkey kidney cells was investigated by using the methionine analogue l-ethionine. In the presence of 2.0 x 10(-3) and 4.0 x 10(-3) moles ethionine, the growth of HeLa and monkey kidney cells was significantly inhibited. Under the same experimental conditions, ethionine had no significant effect on the biosynthesis of two strains of poliovirus (Mahoney and Lansing) in HeLa cells, whereas in primary monkey kidney cells, it markedly inhibited the biosynthesis of the Lansing strain of poliovirus. HeLa cells partly depleted of their intracellular amino acids did not change the rate of viral biosynthesis. The inhibitory effect of ethionine on cell growth and viral biosynthesis was reversed by addition of an excess of l-methionine.