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Previous studies have shown that cementum-bound endotoxin or endotoxin-like products from untreated periodontally involved teeth depress the proliferation of, and have direct toxic effects on, cultured fibroblasts. The purpose of this investigation was to study the effect(s) of ascorbate and several other compounds on the toxicity of commercially prepared endotoxin. The 3T6 fibroblasts were grown in Dulbecco—Vogt modification of Eagles' medium containing 10% heat-inactivated calf serum. Replicate cultures were prepared and grown for 5 days. The proliferation of endotoxin-challenged cells was monitored in the presence of either l-ascorbate, d-isoascorbate, dehydroascorbate, carbonylcyanide m-chlorophenylhydrazone, or cycloheximide. Under very specific conditions, ascorbate reversed the endotoxin-mediated depression of cellular proliferation. An uncoupler of oxidative phosphorylation, carbonylcyanide m-chlorophenylhydrazone also effectively reversed the effect of endotoxin while cycloheximide had no...

It has been suggested earlier that the local deficiency of ascorbic acid in tissues could be responsible for development of various angiopathies in diabetes. Hyperglycemia is one of the factors which could contribute considerably to the development of local ascorbic acid deficiency. Therefore, the effect of glucose on uptake of L-[1-14C] ascorbic acid by fibroblasts was studied in vitro. The data clearly show that ascorbic acid uptake is inhibited instantly by glucose in a concentration dependent fashion. The results support the contention that local ascorbic acid deficiency in tissues could be a natural consequence of hyperglycemia of whatever cause. The rate of ascorbic acid uptake under various conditions suggests that additional supplements of ascorbic acid might be helpful to individuals in averting deleterious effects of hyperglycemia on tissue ascorbic acid supply.

Polymyxin B sulfate treatment induced an increase in calcium influx in mouse fibroblasts (3T6) and normal human skin fibroblasts. This increase in calcium influx occurred in a dose- and time-dependent fashion and was dependent on pH but independent of the electrochemical gradient of calcium across the plasma membrane. This effect was prevented when cycloheximide (20 micrograms/ml) was added with polymyxin B sulfate. Addition of actinomycin D (2 micrograms/ml) also remarkably reduced this effect. In view of these findings, it is possible that polymyxin B sulfate therapy-induced side effects, such as neuromuscular blockade and kidney dysfunction, are conditional and may be due to an increase in calcium influx.

The role of cell density and pH on calcium influx was studied in normal and endotoxin-challenged cultured 3T6 fibroblasts. In normal fibroblasts, at low cell densities, there was no marked difference in calcium influx at pH 6.6, 7.4, and 7.8, whereas at high cell densities, the calcium influx was markedly higher at pH 6.6 as compared to that at pH 7.8. Endotoxin treatment for 4 hr at low cell density and in alkaline pH (7.4-7.8) increased calcium influx in a dose-dependent manner. In contrast, at high cell density and low pH (6.6), endotoxin treatment markedly decreased calcium influx in a dose- and time-dependent manner. These endotoxin-induced changes in calcium influx were not fully compensated by altered calcium efflux because total calcium content of the cells was found to be altered. The efficacy of the endotoxin varied depending on the bacterial source of the endotoxin and the method of purification. There was a relationship between the effect of different endotoxins on the increase in calcium influx and the inhibition of cell proliferation. Endotoxin did not decrease, but slightly increased cell proliferation when added to high cell density cultures even at a concentration of 200 micrograms/ml.

An hypothesis is proposed relating the possible role of vitamin deficiency as an etiologic factor contributing to periodontal disease in diabetes. The hypothesis is based upon the following: (1) transport of ascorbate across cell membranes may be impaired by glucose, but facilitated by insulin; (2) glucose utilization is significantly accelerated by sublethal concentrations of endotoxin; (3) endotoxin-induced histamine sensitivity of tissue is enhanced by ascorbic deficiency; and (4) ascorbic acid deficiency alters mucosal barrier function. The interrelationship of these factors is discussed.

Perfusion of isolated intestinal loops has been utilized in the management of uremia, although the technique has been limited by an inability to adequately extract creatinine. In a recent study of uremic rats, the addition of sodium deoxycholate (NaDC) to the perfusate increased clearance of sodium and creatinine sevenfold. This investigation was designed to confirm this effect in canines and to determine if increased creatinine excretion can be maintained in the absence of net water flux. Isolated jejunal loops of 38 to 75 cm in length were created in conditioned canines. Following a 2-week recovery period, loops were perfused with control solutions (Na 60 meq/liter, K 8 meq/liter, Cl 65 meq/liter, polyethylene glycol 5 g/liter, adjusted osmolarity 360 mosm/liter), and experimental solutions of comparable composition and osmolarity including 2 mM NaDC. NaDC perfusion increased loop creatinine excretion four- to sevenfold allowing clearances of up to 79 mg of creatinine/hr. This increase was statistically significant (P < 0.01) when compared to control perfusions (control 6.00 ± 5.16 mg/hr vs NaDC 37.54 ± 16.61 mg/hr). No net water flux and minimal electrolyte flux were noted. Longer loops required higher perfusion rates to maximize creatinine excretion; optimal perfusion rates approximated 40 ml/hr/cm. Bile salts significantly increased creatinine secretion from isolated jejunal loops without obligatory electrolyte or water movement. Addition of such local agents may improve the efficiency of intestinal dialysis and allow reconsideration of clinical use.

The effect of bacterial endotoxin on the ascorbic acid uptake by 3T6 fibroblasts was studied. Endotoxin inhibited ascorbic acid uptake by fibroblasts in a dose dependent manner. The inhibition by endotoxin takes place only in the presence of unheated serum; decomplementing serum by heat inactivation at 56 degrees C for 30 minutes eliminates endotoxin's inhibitory effect on ascorbic acid uptake. The effect of endotoxin appears to be instantaneous since the inhibition seen in the cells without any preexposure was similar to the cells preexposed to endotoxin for up to 6 hours. Polymyxin B sulfate which is known to bind the lipid A portion of endotoxin did not reverse the inhibition of ascorbic acid uptake caused by endotoxin.

Endotoxin was found to be nonselective in its cytotoxicity of cellular organelles. All organelles studied displayed some degree of alteration which became more severe as the concentration of endotoxin was increased. The data suggest that the metabolism of the cell may be compromised at concentrations of endotoxin which do not affect cell viability. The problem involved in attempting to determine the actual concentration of endotoxin at the interface between the culture medium and the cell monolayer, in vitro, and the corresponding situation, in vivo, are also discussed.

SummaryThe exposure of 3T6 fibroblasts to the lathyrogen β-aminopropionitrile fumarate caused a reduction in proliferation of the cells but had no effect upon viability. Chemical analysis for the production of collagen as measured by free, peptide, and protein-bound hydroxyproline showed that the lathyrogen exerted a stimulative effect. The lathyrogen-treated cells produced approximately twice the amount of hydroxyproline as did the control cells. The data also indicate that the initial stages of collagen production are not altered and that the depression in proliferation signifies a change in overall metabolism rather than a direct toxic effect.