Lack of an EMF-induced genotoxic effect in the Ames assay.

A few epidemiological studies have linked exposure to electromagnetic fields (EMF) and the incidence of cancer. Since many carcinogens are mutagens in the Ames assay, the purpose of this study was to determine if exposure of four tester strains of Salmonella typhimurium (TA97a, TA98, TA100, and TA102) to EMF would increase their rate of mutation. Parallel plate electrodes and Helmholtz coils were used to create uniform field properties (300 V/in., 0.3 mT). Separate and combined alternating electric and magnetic fields effects were studied at a combined field frequency of 60, 600, and 6000 Hz at room temperature. These fields did not elevate the temperature of the culture plates above room temperature, Petri dishes containing each tester strain in top agar were exposed to an electric field (E), magnetic field (M), combined electric and magnetic field (EM), or no additional field above ambient conditions in the lab (control). Four plates containing each strain were exposed in each condition: two plates had the appropriate positive-control mutagen for each strain included in the top agar and two plates did not. Plates were exposed to either E, M, EM, or control conditions at room temperature for 48 hr. and then incubated an additional 24 hr. at 37 deg. C. The plates containing mutagen in the top agar showed an increased number of colonies consistent with mutagenesis. However, the rate of mutation in the S. typhimurium strains TA97a, TA98, TA100, and TA102 in either the presence or absence of mutagen was not affected by 48 hr. exposure at room temperature to E, M, or EM fields at 60, 600, or 6000 Hz.

Effects of short term exposure to 60 Hz electromagnetic fields on interleukin 1 and interleukin 6 production by peritoneal exudate cells.

The relationship between exposure to electromagnetic fields (EMF) and human health is of increasing interest. Exposure to EMF has been linked to leukemia and brain tumors in some but not all epidemiological studies. The effects of separate and combined alternating electric and magnetic fields on interleukin 1 (IL-1) and interleukin 6 (IL-6) production were measured in this study. Helmholtz coils and parallel plate electrodes were used to create uniform field characteristics (300 V/in., 0.3 mT). Effects were studied at a combined field frequency of 60 Hz. This frequency did not elevate culture temperatures above ambient room temperature. Murine thioglycollate-elicited peritoneal exudate cells (PEC) were exposed to an electric field (E), magnetic field (M), combined electric and magnetic field (EM), or no field (control). Three samples of PEC from each mouse were cultured with lipopolysaccharide in each field. Using commercial ELISA kits, supernatants of cell cultures were tested in duplicate after 24 hours of exposure for IL-1 alpha levels and after 48 hours of exposure for IL-6 levels. Results were evaluated using one-way analysis of variance (ANOVA). As a group, IL-1 production by the PEC from five mice and IL-6 production by the PEC from nine mice were unaffected by electric, magnetic, or combined electric and magnetic fields. Results from these experiments indicate that the 24-hour exposure to 60 Hz electric, magnetic, or combined electromagnetic fields had no effect on IL-1 production. Forty-eight hours of exposure to the same fields did not affect IL-6 production.

Effect of sodium ascorbate on the chemiluminescent response of murine peritoneal exudate cells.

Vitamin C is an effective antioxidant that neutralizes reactive oxygen radicals. The purpose of this study was to determine if sodium ascorbate would neutralize the reactive oxygen products generated during the respiratory burst of thioglycollate-elicited murine peritoneal exudate cells (PEC). In vitro and in vivo studies were done. Cells treated in vitro showed a significant, dose-dependent reduction in chemiluminescence (CL) after activation with opsonized zymosan. Higher concentrations of sodium ascorbate (24.2 mM) produced a significantly greater reduction in CL than did lower concentrations (0.242 mM). This range of sodium ascorbate concentrations overlaps those found in normal leukocytes (1-4 mM). Sodium ascorbate at physiological plasma concentrations (0.09 mM) did not reduce CL. Cells incubated with 500 mM sodium ascorbate in vitro and then washed once prior to zymosan activation also showed a significant reduction in CL. In contrast, PEC harvested from mice treated in vivo with sodium ascorbate (one or five daily doses of 1.0 M sodium ascorbate, 0.01 ml/g body weight) did not show a reduction in CL. This concentration of sodium ascorbate represents a dose that is 2310 times greater than the Recommended Dietary Allowance (RDA). These studies show that physiological doses of sodium ascorbate can quench CL in vitro, but even large doses of sodium ascorbate administered in vivo do not affect the CL of harvested murine PEC.

In vivo and in vitro studies on the effects of vitamin A on the chemiluminescent response of murine peritoneal exudate cells.

Retinoids are known to enhance macrophage function and enhance bacterial clearance during experimental infection. The purpose of these experiments was to determine if one indicator of macrophage activation, chemiluminescence (CL), was enhanced by retinoids. Peritoneal exudate cells (PEC) harvested from mice injected intraperitoneally for 5 days with retinol palmitate (25, 125, or 250 U/g body wt./d) showed significantly enhanced chemiluminescence (CL) when exposed to opsonized zymosan. A direct dose-response effect was observed, in that the more retinol palmitate was injected, the more CL was observed. The same dose of retinol palmitate injected subcutaneously did not result in enhanced CL. In vitro incubation of murine PEC with physiological concentrations of retinol palmitate or retinoic acid for 1, 6 or 24 h did not result in enhanced CL. The reasons for the effect of the route of administration of retinol palmitate are unknown, but may include poor absorption from the site of subcutaneous injection or an adjuvant effect when injected intraperitoneally due to the particulate nature of the water-dispersible retinol palmitate preparation.

Effects of prenatal and postnatal exposure to vitamin A on the development of the murine immune system.

Vitamin A and its derivatives are known to enhance the immune system and affect embryogenesis. In this study, five daily subcutaneous injections of retinol palmitate (0.001 mg/kg body weight) were administered to eight female SW mice before mating. Six more weekly injections of retinol palmitate were given during pregnancy and lactation. Eight controls were similarly treated with saline. Four of the eight vitamin-A-treated females had litters, whereas seven of the eight saline-treated females had litters. Resultant litters did not differ in size or appearance. At 12 weeks of age, serum IgM and IgG1 levels were significantly higher in the progeny of vitamin-A-treated mothers before but not after immunization with a test antigen, sheep red blood cells (SRBC). This difference was not seen when other progeny were tested at the age of one year. Anti-SRBC titers did not differ in the two groups of progeny at the age of 12 weeks or one year. One-year-old progeny of vitamin-A-treated mothers weighed significantly more than did control progeny; significant enlargement of the heart, spleen, and kidneys was observed. However, organ-to-body-weight ratios did not differ significantly. In a separate experiment, adult female mice treated with varying doses of vitamin A (five daily doses of 0.0001, 0.0005, or 0.001 mg/kg body weight) showed a dose-dependent reduction of serum IgG1 and hematocrits, but no change in serum IgM levels or leukocyte counts. Resting untreated mice had IgM levels which were one-half those seen in saline-treated controls. These studies indicate that large doses of vitamin A can affect some aspects of the developing and mature murine immune system.

Immunotoxicology studies on octoxynol-9 and nonoxynol-9 in mice.

In a double-blind study, mice were injected intraperitoneally with 0.2 ml 0.2% octoxynol-9 (O-9), 0.2 ml 0.2% nonoxynol-9 (N-9), or 0.2 ml saline (control) daily for 24 days. Another control group received no treatment. All mice were immunized twice with sheep red blood cells (SRBC) and bled by caudal incision. Mice receiving N-9 lost weight (P less than 0.02), had smaller livers (P less than 0.05), and showed enlarged spleens (P less than 0.05). The N-9-treated mice did not differ from either control group in the primary or secondary anti-SRBC responses, leukocyte (WBC) counts, or in the sizes of the kidneys, hearts, lungs, or thymuses. Mice receiving O-9 showed no significant differences from either control group in any of these tests. Serum immunoglobulin M (IgM) and immunoglobulin G (IgG) levels were similar in mice treated with O-9, N-9, or saline. All 3 groups had higher levels of both classes of immunoglobulins on day 16 than did untreated controls. This study shows that O-9, given to mice in doses 3 times that used by humans, is nontoxic, whereas the same dose of N-9 has minor deleterious effects.

Dimethyl sulfoxide: lack of suppression of the humoral immune response in mice.

Mice were injected intraperitoneally (i.p.) with 100% dimethyl sulfoxide (DMSO) or sterile saline (controls) for 36 days. The mice received 0.05 ml daily for one week, 0.025 ml every other day for the second week (because the DMSO-treated mice appeared weak), and 0.05 ml daily for 3 more weeks. All mice were immunized twice with sheep red blood cells (days 13 and 24), and bled twice by caudal incision (days 20 and 29). Hematocrits were significantly decreased (P less than or equal to 0.002) but still within the normal range. The primary and secondary antibody response to sheep red blood cells (SRBC), leukocyte counts, body weight, and the size of the heart, lungs, spleen, thymus, and kidneys were not affected. DMSO treatment resulted in significant liver enlargement (P = 0.02). It is concluded that this dose of DMSO is not deleterious to the humoral immune response in mice responding to a new antigen.

Effects of deceleration on the humoral antibody response in rats.

The purpose of this experiment was to determine the effects of hyper-gravity (hyper-G) followed by a return to normal gravity (deceleration) on the immune system of rats. Hyper-G was simulated by chronic centrifugation. Eighteen 35-d-old male rats were divided into three groups of six rats each. Two groups were exposed to 2.1 G and 3.1 G, respectively, for 28 d. The third group served as 1.0 G controls. Rats were removed from the centrifuge on day 29. All rats were immunized with sheep red blood cells (SRBC) on days 29, 42, and 57; rats were bled on days 36, 47, and 62. Hematocrits and anti-SRBC titers were determined each day the rats were bled. White blood cell (WBC) counts were determined on day 47. On day 63, all rats were sacrificed and organ:body mass ratios obtained for a number of organs. Centrifuged rats ate and gained significantly less. The organ:body mass ratios for the adrenal glands, kidneys, lungs, heart, and thymus were unaffected by deceleration. Although marginally significant (p less than 0.05) decreased organ:body mass ratios were found for the liver and spleen in the 3.1 G group, this effect may not be real, due to the small number of rats used. No significant differences were found in hematocrits, WBC counts, or anti-SRBC titers. These experiments indicate that deceleration does not adversely affect these particular aspects of the immune system. However, they do not preclude the possibility that other aspects of the immune system, such as interferon or complement, may be affected.

Methyldopa: effects on the murine immune system.

Mice were treated for 7 weeks with doses of methyldopa somewhat exceeding those given to man, and mixed immunotoxic effects were observed. Daily subcutaneous injections of 5 mg (in 0.1 ml) methyldopa or saline (0.1 ml) did not generally alter body weights, except on day 19, when the methyldopa-treated mice weighed significantly less. During treatment, all mice were immunized twice with sheep red blood cells (SRBC) and bled four times. Anti-SRBC titers were not affected by methyldopa treatment, but leukocyte counts were dramatically decreased, and hematocrits to a lesser degree. Although in methyldopa-treated mice spleen and kidneys were increased in size, liver, lung, heart, and thymus size was not affected. These results are discussed in the context of other studies on the mode of action of methyldopa in eliciting an autoimmune anemia in man treated therapeutically with this drug.

The immune system: effects of hypergravity and hypogravity.

The force of gravity has been inescapable until only the last few decades. Space programs conducted by several nations now make possible the study of hypergravity and hypogravity in a variety of scientific areas. Although much work has focused on the physiological aspects of gravity, its effects on the immune system are only beginning to be appreciated. An understanding of these effects is not only of theoretical interest, but important in predicting the health of astronauts exposed to hypergravity and hypogravity. These studies may also help to answer the larger question of how stress affects the immune response.

The unresponsiveness of the immune system of the rat to hypergravity.

The immune response in rats exposed to simulated hypergravity (2.1 G and 3.1 G) by chronic centrifugation was assessed. Rats were immunized with sheep red blood cells (SRBC), either on the day of initial exposure to hypergravity (hyper-G), or after being centrifuged for 28 d and remaining on the centrifuge thereafter. Pair-fed and ad libitum fed noncentrifuged controls were used. Although there were some alterations in leukocyte counts, hyper-G did not systematically affect the primary or secondary anti-SRBC response, hematocrits, or the sizes of the liver, spleen, kidneys, thymus, or adrenal glands. The immune system is thus remarkably homeostatic under hypergravity conditions which do affect other physiologic parameters.

Effect of ethanol on the immune system in mice.

Mice were injected with ethanol (2.5 g/kg body wt., i.p.) 1-3 times daily for 17 days or 3 times daily for 49 days. The primary and secondary antibody titers to sheep red blood cells were determined. In addition, microhematocrits, white blood cell counts, white blood cell differential counts, and organ-to-body-weight ratios were determined. Despite continuous large doses of ethanol, the ethanol-treated mice responded as well as saline-treated controls in all parameters tested. The results of this study are discussed in the context of other studies on the effects of ethanol on the immune system.

Demonstration of gene dosage effects on antigens in the Duffy, Ss, and Rh systems using an enzyme-linked immunosorbent assay.

Using a recently developed enzyme-linked immunosorbent assay, gene dosage effects were demonstrated for the Duffy, Ss, and Rh red blood cell antigen systems. We report a comparison by an antibody-binding assay of the relative amounts of Fya and Fyb antigens on FyaFya, FyaFyb, FybFyb, FyaFyx, and FyxFyx erthrocytes, and of s antigen on cells homozygous and heterozygous for s. The immunosorbent assay also was used to study Rh antigens, and data which had been obtained using radiolabelled antibodies were confirmed.

Effect of simulated weightlessness on the immune system in rats.

Rats suspended in a model system designed to simulate many aspects of weightlessness were immunized with sheep red blood cells. Parameters measured on these and control rats included titers of anti-sheep red blood cell antibodies, serum immunoglobulin levels, spleen and thymus weights, hematocrits, and leukocyte differential counts on peripheral blood. No significant differences were found between test and weight-bearing, harnessed controls; however, the thymuses of animals in both these groups were significantly smaller than untreated cage controls. The lack of an effect of simulated weightlessness on the immune system is an interesting result, and its significance is discussed.

The role of complement in resistance to endogenous and exogenous infection with a common mouse pathogen, Corynebacterium kutscheri.

In mice, the presence or absence of a single complement (C') component, called hc(1), is controlled by two alleles at the Hc locus. The sera of mice which lack this C' component do not manifest C'-mediated immune hemolysis. When challenged with the common mouse pathogen, Corynebacterium kutscheri, mice possessing hemolytic C' fare slightly better than C'-deficient mice. When mice harboring latent C. kutscheri are administered hydrocortisone, which depresses mouse serum C' levels, pseudotuberculosis is activated with equal frequency in mice of both C' types. These data suggest that in at least one situation the presence of the complete hemolytic C' system may be advantageous to the mouse. In contrast, evidence is presented which shows that under normal laboratory conditions, C'-deficient B10.D2 "old line" mice (Hc(0)/Hc(0)) have a survival advantage over C'-positive B10.D2 "new line" mice (Hc(1)/Hic(1)) during the first 3 wk of life. It is therefore concluded that mouse hemolytic C' has a balanced survival value-that is, under one set of conditions it may be advantageous, whereas in another situation, it may be disadvantageous.

Steroids and serum complement in mice: influence of hydrocortisone, diethylstilbestrol, and testosterone.

Hydrocortisone depresses hemnolytic complement in male and female mice. Testosterone causes increase of serum complement in female mice, and diethylstilbestrol causes decrease of serum complement in male mice, in each instance to activities approximating those found normally in the opposite sex. Male and female sex hormones have no effect, in the doses used, on the serum complement of male and female mice respectively.