Elliott J. Greene

In vitro mutagenicity and cell transformation screening of phenylglycidyl ether

Phenylglycidyl ether (1,2-epoxy-3-phenoxy propane) (PGE) was tested for genetic activity in bacterial and mammalian tests. It was active in the Salmonella/microsome mutagenicity test. Concentration-dependent mutagenicity was demonstrated in S. typhimurium strains TA1535 and TA100 with and without rat S9, but not in strains TA98, TA1537, or TA1538. These results suggest PGE, is a direct-acting mutagen causing base substitutions. Phenylglycidyl ether did not induce 6-thioguanine-resistant mutants of Chinese hamster ovary cells, with or without rat S9, and with or without serum in the medium. Dose-dependent enhancement of SA7 virus transformation of primary hamster embryo cells was observed at concentrations of 1.6 microgram/ml and higher. In addition, this compound was able to chemically transform secondary hamster embryo cells at concentrations of 6.2 micrograms/ml and higher. At a dose of 2500 mg/kg p.o., PGE was active in the host-mediated assay using C57B1/6 X C3H mice and S. typhimurium strain TA1535. This activity represented a positive response in 2 of 5 animals tested. Murine testicular DNA synthesis was not inhibited by oral administration of PGE at 500 mg/kg.

In vitro evidence indicating a role for the Fc region of IgG in the mechanism for the long-term maintenance and regulation of antibody levels in vivo

Abstract The synthesis of anti-human serum albumin (HSA) antibody was induced spontaneously in cell cultures prepared from the draining lymph nodes of rabbits immunized months earlier with polymerized HSA. Serum from HSA-immunized rabbits suppressed this response. Removal of specific antibody from immune serum eliminated suppression and the addition of specific IgG restored suppression, indicating that the feedback phenomenon may be explained by an effect of specific IgG antibody. Fab and F(ab′) 2 fragments masked antigen as effectively as IgG; however, they were markedly inferior to IgG in mediating suppression. Furthermore, Fab competed with IgG and interfered with IgG mediated suppression. The addition of small amounts of antigen to antibody-suppressed cultures induced an antibody response. The level of induction was proportional to the antigen-antibody ratio. However, 80 to 100 times the antibody concentration needed to mask all antigenic determinants was needed in order to eliminate induction of antibody synthesis. High concentrations of antigen-antibody complexes at equivalence also suppressed the spontaneous response. This suppression was similar to antibody mediated suppression at the spontaneous response in that the Fc region of IgG was required.

Paradoxical effects of piperonyl butoxide on the kinetics of mouse liver microsomal enzyme activity.

Abstract The kinetics of the effects of piperonyl butoxide on mouse liver aminopyrine demethylase, aniline hydroxylase and biphenyl hydroxylases were investigated. Piperonyl butoxide competitively inhibited aminopyrine demethylase in vitro and in vivo, and aniline hydroxylase in vitro; however, in vivo inhibition of aniline hydroxylase was uncompetitive. Contrastingly, in vitro piperonyl butoxide noncompetitively inhibited biphenyl-4-hydroxylase, while competitively stimulating biphenyl-2-hydroxylase. Sulfoxide, a structurally related methylenedioxyphenyl derivative, also noncompetitively inhibited biphenyl-4-hydroxylase and competitively stimulated biphenyl-2-hydroxylase. Furthermore, when microsomal enzyme preparations from control and piperonyl butoxide-treated mice were mixed, aminopyrine demethylase and aniline hydroxylase activities were the same as the control alone, indicating that the inhibitory factors were enzyme bound.

A radioimmunoassay for human antibody specific for microbial antigens.

A simple and sensitive method for detecting and quantitating antibody specific for microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and --log2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri, and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens.

In vitro cell transformation screening of 4 toluene diamine isomers.

2,4-, 2,5-, 2,6- and 3,4-Toluene diamine (TDA) were tested for their ability to enhance the transformation of primary hamster embryo cells (HEC) by Simian adenovirus 7 (SA7) when administered either prior to or after virus inoculation and for their ability to transform secondary HEC. 2,4-TDA was inactive when given prior to SA7, but active if given after. 2,5-TDA was active in both protocols. 2,6-TDA was marginally active if administered before virus and was the most active of the isomers when administered after virus. 3,4-TDA was the most active compound when added prior to SA7, and was also active when given after virus. All the isomers were capable of producing good dose--responses and absolute increases in the number of virus-transformed foci per dish in one or both of the experimental regimens. Each isomer chemically transformed secondary HEC but good dose--responses were rare, and none of the chemicals were active in more than 50% of the 5 or 6 separate tests performed on each.

In vitro mutagenicity and cell-transformation screening of N-(2,3-epoxy-propyl)-phthalimide.

N-(2,3-Epoxy-propyl)-phthalimide (EPP) was tested for genetic activity in the Salmonella/microsome mutagenicity test. Concentration-dependent mutagenicity was demonstrated in S. typhimurium strains TA1535, TA1537 and TA100 with and without rat S9. It was inactive in strain TA1538, and active without rat S9 in TA98 at the high dose. EPP induced 6-thioguanine-resistant mutants of Chinese hamster ovary cells in the absence of an exogenous activating system. EPP produced dose-dependent enhancement of SA7 virus transformation of primary hamster-embryo cells, and transformed secondary hamster-embryo cells in a non-dose-related fashion. At a dose of 5 g/kg p.o. or i.m., EPP was inactive in the host-mediated assay using C57Bl/6XC3H mice and S. typhimurium strain TA1535. Murine testicular DNA synthesis was not inhibited by oral administration of EPP at 1000 mg/kg.

A competition assay for antibody avidity

Abstract A new assay for antibody avidity was developed. This assay depended upon the competition between radiolabeled test antibody and unlabeled standard antibody for a limited number of sites on an immunoabsorbent. Analysis of the binding curves yielded a value which was inversely proportional to the relative avidity of the radio-labeled test vs the unlabeled standard antibody. This avidity value was consistent with a theoretical mathematical model for the system, and was accurate and reproducible. This assay was then used to determine the effects of antigen dosage and antibody feedback upon the avidity of antibody produced in an in vitro culture system. The in vitro findings confirmed the results of numerous in vivo studies showing a decrease in avidity with increasing antigen dose and an increase in avidity under conditions of antibody-mediated suppression.

Effects of specific antigen and specific antibody on the kinetics of in vitro antibody production

Abstract An in vitro model system was used to probe the regulation of an ongoing antibody response. This system employed lymphocyte cell cultures prepared from the draining lymph nodes of rabbits immunized with glutaraldehyde-polymerized or heat-aggregated human serum albumin. The kinetics of specific antibody production in response to soluble antigen given at culture initiation varied among cultures obtained from different animals, often resulting in two distinct peaks of antibody production. Dose-response studies involving antigen added at culture initiation revealed that the overall kinetic pattern was unaffected by antigen dose. However, doses of antigen which suppressed the amount of antibody produced early in culture (96–144 hr) did not suppress, and usually enhanced the amount produced late in culture (192–240 hr). The addition of a second dose of antigen 96 hr after culture initiation was sometimes capable of increasing late antibody production over untreated controls. Dose-response studies for antigen added at 96 hr revealed an apparent 100-fold or greater increase in antigen sensitivity of putative precursors of late antibody-forming cells over that of precursors of early antibody-forming cells. The addition of specific antibody at culture initiation was found to inhibit the late antibody response less than the early response. When antibody was added shortly after antigen had been removed, the early response retained susceptibility to its suppressive effects, while the late response was no longer inhibited. The late response of cultures suppressed by specific antibody given at culture initiation showed the same dose response to antigen given at 96 hr, relative to nonrestimulated controls, as nonsuppressed cultures. Specific antibody given at 96 hr failed to suppress and sometimes enhanced the late response. The combined effects of specific antigen and antibody added at 96 hr in culture were additive, thereby indicating that the two effects were independent of each other. These results were interpreted to suggest that two distinct precursors of antibody-forming cells were present in this in vitro cell culture system.

Acute toxicity of N-(2,3-epoxy-propyl)-phthalimide

N-(2,3-Epoxy-propyl)-phthalimide (EPP) was tested for its oral LD50, its ability to cause ocular and dermal irritation in rabbits, and its ability to sensitize guinea pigs. The single-dose rat oral LD50 was found to be 4.7 g/kg. This chemical was slightly irritating to the rabbit skin, moderately irritating to the rabbit eye without a washout, and mildly, transiently irritating to the rabbit eye with a washout 4 sec after instillation. It did not induce a sensitization reaction in guinea pigs after 10 intracutaneous injections, although this regimen did induce a very mild irritation evident after the eighth injection. These data were interpreted to indicate that EPP did not represent a substantial acute toxic risk.