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In this study, we have analysed the effect of ethanol and phosphatidylethanol, a unique phospholipid formed only in the presence of ethanol, on the binding of [3H]inositol 1,4,5-trisphosphate to rat cerebellar membranes. Rats were intraperitoneally injected daily with 3 g of ethanol/kg body weight for different periods of time. Repeated administration of ethanol induced a reduction in the binding capacity (Bmax) without affecting the affinity constant (Kd). A significant 32% reduction was observed after 21 days of exposure (from control Bmax values of 25 +/- 3 pmol/mg and Kd values of 9 +/- 2 nM). In an in-vitro assay, phosphatidylethanol (500 microM) and phosphatidic acid (500 microM, but no other phospholipids tested, induced a reduction in Bmax (39% and 43%, respectively). The observed effect displayed by phosphatidylethanol was not due to its degradation to phosphatidic acid or other phospholipids. The results emphasize the importance of examining phosphatidylethanol (PEth) as a possible mediator of the effects of ethanol on cellular processes. However, the role of PEth in the observed effect of long-term ethanol exposure still needs further consideration.

Dimethyl morpholinophosphoramidate (DMMPA), an organophosphate, caused leukemia in male and female Fischer 344/N rats. DMMPA was administered in corn oil by oral intubation to groups of 50 male and 50 female rats at 0, 150, 300, or 600 mg/kg body weight, five times per week for 2 years. B6C3F1 mice were given 0, 150 (males only), 300, and 600 (females only) mg/kg body weight under the same schedule. DMMPA induced a dose-related enhancement in the incidence of mononuclear cell leukemia in rats--males: controls = 14/50, 150 mg group = 21/50; 300 mg group = 19/50; 600 mg group = 25/50; females: controls = 9/50, 150 mg group = 13/50; 300 mg group = 12/49; 600 mg group = 18/50. Survival-adjusted rates strengthen the DMMPA effect: males--31%, 50%, 47%, and 63%; females--20%, 32%, 30%, 50%. Latent periods for mononuclear cell leukemia development in exposed rats were not shortened compared to controls. No carcinogenic effects in mice were detected. DMMPA was not mutagenic in Salmonella, was mutagenic for mouse lymphoma cells, and induced both chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells.

The ability of 2 recent ether-lipid derivatives, aza-phospholipids BN52205 and BN52211, to induce apoptosis in different leukemia cell lines was investigated using I-octadecyl-2-methyl-rac-glycero-3- phosphocholine (ET-18-OCH3) as a positive control. HL60, K562, Molt-4 and U937 cells were exposed for 24 hr to 20 microM of drug. The 2 aza-derivatives were as cytotoxic as ET-18-OCH3: BN52205 and BN52211 selectively induced apoptotic death in HL60, Molt-4 and U937 cells, but not in the K562-resistant cell line. Around 50% of DNA was fragmented in HL60 cells after exposure to the aza-derivatives, and 34% and 20% of DNA was fragmented in Molt-4 and U937 cells respectively. Similar results were obtained when cells were exposed to ET-18-OCH3. Our data confirm that ether lipids induce apoptosis in a variety of human leukemic cells, providing a possible explanation for their selectivity and mechanism of action.

Membrane-interactive ether lipids (EL) exert toxic and antiproliferative effects on cancer cells in vitro. They appear to be selectively more toxic to cancer cells than to normal cells and thus they are ideal candidates for bone-marrow purging procedures. However, no conclusive explanation has yet been provided for this property. We now present some data indicating that the cholesterol concentration in the incubation medium modulates EL toxicity against the HL60 leukemic cell line in vitro. Furthermore, model membranes richer in cholesterol take up EL more slowly, and cell cholesterol enrichment of HL60 cells counteracts EL biophysical membrane interaction, but not toxicity, in our experimental model. However, the K562 cell line, a leukemia line less sensitive to EL toxic action, has higher levels of cell cholesterol. Our data provide evidence to explain differences in sensitivity to EL among different cell types and contribute to the understanding of the mechanism of action of EL.

Lipoteichoic acid (LTA) was prepared from type III group B streptococci and administered by topical oral application or intravenous or intratracheal injection in weanling and adult white New Zealand rabbits. Tritiated [3H]LTA in tissues and body fluids was measured by scintillation spectrometry. Five minutes to 120 h after intravenous injection of 10 mg (17 x 10(6) dpm) of [3H]LTA, none was present in blood. Combined urine and fecal excretion peaked at 24 h and decreased over 5 days. There was no effect on collagen-induced platelet aggregation. [3H]LTA concentrations were greatest in colon, bone, stomach, and skin 1 day after intravenous injection. After a 5-mg oral dose (8.5 x 10(6) dpm) in an adult animal, fecal excretion peaked at 24 h and decreased after 4 days. No systemic absorption was noted. No [3H]LTA was found in any of seven tissues examined at autopsy 3 days after 1 to 5 mg/kg oral doses in weanling animals with normal or traumatized buccal mucosa. No effect was noted on platelet aggregation or serum complement, there was no increase in the incidence of nephrocalcinosis and the buccal mucosa remained histologically normal. Intratracheal injection of 0.5 to 2.5 mg/kg of LTA resulted in no tachypnea or alteration in blood gases. All animals remained healthy after LTA administration. The absence of toxicity and absorption in animals suggests that studies could be performed in humans to evaluate the safety and efficacy of oral LTA.

The toxicity of lipoteichoic acid (LTA) from Streptococcus pyogenes type 12 was investigated by using mouse fibroblasts in culture in the absence of serum. Morphologically, while low concentrations of LTA elicited a subtle effect characterized by progressive cellular degeneration with practically no release of protein, larger concentrations (greater than 50 micrograms/ml) of this amphiphile resulted in rapid death of cell monolayers. Metabolic studies utilized a concentration of LTA (17.5 micrograms/ml) which caused the smallest change in cell morphology in the least number of mouse fibroblast cells per monolayer. Under these conditions, cell monolayers showed an increase of 450% in their content of collagenous protein after exposure to LTA. However, the amount of such material secreted remained unchanged. Also, changes in the type of collagenous protein formed were observed after exposure to LTA. Collagenous protein accumulating intracellularly was found to be practically hydroxyproline-free. However, collagenous protein secreted by this cell line showed a significantly reduced content of hydroxyproline as compared with control cells unexposed to this coccal membrane component. Column chromatographic studies confirmed that the collagenous protein secreted by monolayers exposed to LTA was defective (under hydroxylated). It was concluded that LTA does not affect the amount of collagenous protein secreted. However, it does increase the amount of this protein formed and retained by this cell line as well as causing a reduction in the hydroxylation of proline in both intracellular and secreted collagenous material. A possible relationship between abnormal basement membrane morphology and disturbed collagen synthesis in post-streptococcal glomerulonephritis as related to LTA is discussed.

The ability of LTA of Streptococcus pyogenes to stimulate cell division or to kill tissue culture cells derived from human heart was investigated. Initial studies indicated that at low concentrations, ranging from 0.01 to 1.0 micrograms/ml, LTA stimulated cell division, whereas at higher concentrations, ranging from 10 to 1000 micrograms/ml, it killed the cells. Deacylated lipoteichoic acid, which lacked cell membrane binding activity, similarly stimulated or killed the heart cells depending on the concentration added to the tissue cultures. Fractionation of LTA after mild ammonia hydrolysis yielded a polyglycerophosphate and polar lipid fraction, both of which retained the glucose from the glycolipid moiety of the LTA molecule, and a neutral lipid fraction that was devoid of phosphorus or glucose. Toxic activity was present only in the fractions containing glucose. Oxidation of LTA or its deacylated derivative with sodium metaperiodate reduced the assayable glucose content without destroying the polyglycerophosphate backbone and resulted in a parallel loss of cytotoxicity, strongly suggesting that the glucose moieties of S. pyogenes LTA must be intact for the toxic activity against human heart cells to be expressed.

The morphology and pathology of cultured mouse glomeruli were examined at the cellular and subcellular levels after infection with a physiological isotonic L-form of Streptococcus pyogenes type 12 or exposure to streptococcal lipoteichoic acid. These changes, as viewed by light microscopy, were identical regardless of the method used to induce glomerular cytotoxicity. They were characterized by an initial reduction in the outgrowth of cells, some cellular granulation, and later, destruction of the confluent monolayer. Once initiated, cytotoxicity could not be reversed by refeedings, and complete glomerular destruction resulted after 2 weeks. Electron microscope studies revealed that the basement membrane of intact glomeruli exposed to streptococcal lipoteichoic acid had become greatly thickened (two- to fourfold) and electron dense. Our recent biochemical findings have shown that streptococcal lipoteichoic acid increases the amount of collagen formed and retained by mouse fibroblasts in tissue culture as well as causing a reduction in the hydroxylation of proline in both intracellular and secreted collagenous material (Leon and Panos, Infect. Immun. 40:785-794, 1983). These results, together with the present findings, suggest that the thickening of the glomerular basement membrane may be due to defective collagen biosynthesis as a result of streptococcal lipoteichoic acid. The use of cultured glomeruli as a model system for studying the earliest basement membrane alterations in the absence of an immune response as a result of streptococcal lipoteichoic acid is suggested.

The inhibition of 3H-thymidine incorporation into DNA of L1210 cells in culture by liposomes was used as an index of liposome toxicity. Inhibition of 3H-thymidine incorporation appeared to be dose dependent for some lipid compositions tested. The commonly used neutral lipids, phosphatidylcholine and cholesterol did not appear to inhibit 3H-thymidine incorporation. Phosphatidic acid, an adjunct for preparing anionic liposomes, appeared to be non-toxic compared to phosphatidylserine and dicetylphosphate (alternative adjuncts for preparing anionic liposomes). Stearylamine, a synthetic lipid which continued to dominate the preparation of cationic liposome was the most toxic of the lipids tested.