Joseph D. Irr

Design of a statistical method for the analysis of mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells

Mutagenesis data collected in the mammalian cell CHO/HGPRT assay were analyzed to study the distribution of the experimental errors associated with the test. The data neither followed the widely assumed Poisson distribution nor satisfied the usual statistical assumptions of normality and homogeneous variance of experimental errors. We transformed the data by using the power formula Y = (X + A) gamma where X is the observed mutation frequency, Y is the transformed frequency, and A and gamma are constants determined by the procedure of Box and Cox. Setting A = 1 and gamma = 0.15 we produced transformed values for which the assumptions of homogeneous variance and normal distribution were satisfied. This transformation enables one to properly use Students t-test and dose-response analysis of variance to analyze CHO/HGPRT results. The experimental design for CHO/HGPRT mutagenesis assays is also discussed.

A procedure for the statistical evaluation of Ames Salmonella assay results comparison of results among 4 laboratories

Ames Salmonella test data collected in our laboratory and 3 National Cancer Institute contract laboratories were analyzed to study the distribution of experimental errors associated with the test. It is shown that the Poisson distribution is not appropriate, and that the power transformation model Y = (revertants/plate)lambda, with lambda = 0.2 as estimated by the methods of Box and Cox, produced a measurement scale on which the experimental errors could be adequately described by a normal (Gaussian) distribution with a constant variance. The modeling procedure enables one to properly use analysis of variance, regression analysis, and Students t test to analyze Ames Salmonella test results, and well-known statistical quality control procedures to monitor laboratory performance. The method detects weak mutagenic activity and measures the amount and uncertainty of the increase in revertants/plate. The development of the power transformation model is discussed and examples of its use in the interpretation of Ames Salmonella assay results are included.

A statistical method for analysis of mouse lymphoma L5178Y cell TK locus forward mutation assay: Comparison of results among three laboratories

Analysis of data from our laboratory and that of two National Cancer Institute contractor laboratories indicate the random variation in the results of the mouse lymphoma L5178Y cell TK locus mutation assay can be adequately described by a lognormal distribution. This indicates that transformation of mutant frequencies to logarithms enables one to properly use well-known statistical techniques such as analysis of variance, regression analysis, and Students t-test for the interpretation of data from this assay. The consistency of the lognormal distribution among laboratories is demonstrated. Three examples which illustrate the mechanics and interpretation of the proposed methodology are included. It is concluded that the method is effective in identifying weak mutagens as well as enabling the user to compute the uncertainty associated with an observed increase in mutagenic activity.

Expansion of lymphokine-activated killer cells for clinical use utilizing a novel culture device

Two major problems encountered in the application of lymphokine-activated killer (LAK) cell therapy in man are the massive culture volumes required for LAK cell induction and the paucity of LAK cells available for administration (human doses are less than or equal to 10% of effective murine LAK cell doses). We have, therefore, developed and tested a plastic porous culture device, Sclair plastic bags (E.I. DuPont De Nemours Co.), that can be utilized at virtually any volume and does not require rotation for optimal use. Normal or patient lymphocytes were cultured in the device or in plastic 16 mm wells at 1-20 X 10(6)/ml RPMI 10% human sera with 1500 pM interleukin-2 for 4 days: LAK cell activity did not decline despite high cell densities. The device was equal to the 16 mm wells in induction of normal donor and patient LAK cell activity when either autologous fresh tumor or Raji targets were used. In a non-therapeutic clinical evaluation we isolated and stored in liquid nitrogen autologous tumor cells from 11 patients with cancer. 2-6 weeks post-operatively lymphocytes and mononuclear cells from these patients and paired normal donors were obtained and LAK cells were induced in Sclair bags or standard culture wells. Autologous patient LAK cell activity and normal donor LAK cell activity against patients tumor cells were equivalent in the Sclair culture device and culture well system. Lymphocyte recovery and [3H]thymidine incorporation were also similar. Subsequently, we developed an expansion scheme utilizing the device in which cell density was maintained at optimal levels by changing media and reducing cell concentration after 6, 10 and 14 days of culture. We were able to expand LAK cell number 5-10-fold with no loss of LAK cell activity in this time frame utilizing both normal and patient cells. In this system plasma and sera were equivalent in their capacity to support LAK cell expansion but less than 10% plasma or sera supported suboptimal activation. Thus, we have developed a practical system to augment the number of LAK cells available for human LAK cell therapy and simultaneously reduce the complexity and volume of the induction system.

An evaluation of epidemiological methods of monitoring human populations for increased genetic risk

Abstract Nine epidemiological methods presently available to study humans for increased genetic risk were reviewed. None of the methods directly associated with mutational endpoints were of sufficient power to be practical for monitoring groups of individuals exposed to potentially mutagenic chemicals. The most promising methods found were those which indirectly evaluate mutagenic effects. These were methods for studying delayed or decreased fertility, and fetal wastage. Significant findings in such studies in the future may reveal that chemically induced mutations are occurring in humans.