E

DBA/2N.XHZ 0.0002

BALB/cALIZ

6 12 18 24 30 36 Mean mutant rate (x 10"5)

Fig. 3. Mean mutant frequencies (± standard deviations) in the spleens of PCT-resistant D2.ALIZ mice and PCT-susceptible B/c./.LIZ mice. Experimental groups consisted of four to six mice. A total of 3.11 x 105 plaques was screened on average in each group. The number of blue mutant plaques enumerated in these groups ranged from 25 to 63.

glutathione (GSH) stores. PCT-susceptible B/c mice displayed significant elevations in splenic mutant rates after treatment with pristane or BSO, whereas PCT-resistant D2 mice did not.9 This result is consistent with the view that the genetic susceptibility to PCT development may be associated with elevated levels of oxidative mutagenesis in lymphoid tissues and B cells.10 The hypothesis that B/c B cells may be particularly susceptible to oxidative mutagenesis was further supported by the result of the experiment summarized in Table I. The À LIZ assay was used to determine the mutant rates in B/c.ÀLIZ spleen cells after separation by magnetic cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) into B220+ B cells and B220" non-B cells. Comparison with the mutant rates in whole spleens obtained from age-matched B/c. À LIZ mice was also included. Treatment with pristane resulted in an increase in mutant levels in whole spleens by 64.8% (from 5.88 x 10~5 to 9.69 x 10~5). The increase was significant (p = 0.0187) in the two-tailed paired t test. Furthermore, although no differences in mutant rates between whole spleens and fractionated B and non-B cells were found in normal B/c mice (Table I, rows 1-3), a significant reduction and elevation in mutant rates was found in non-B cells and B cells, respectively, when fractionated cells obtained from pristane-primed animals were compared with whole spleens of pristane-primed animals (Table I, rows 4-6). These results were interpreted to mean that B/c B cells may indeed experience dysproportionately high amounts of oxidative mutagenesis under conditions of pristane-induced inflammation. D2 B cells may be different in this regard, but this has not yet been demonstrated.

Many groups, including our own, have observed that time-consuming procedures for B cell isolation can result in an unwanted loss of the main cellular antioxidant, GSH, and a drop in the biological activity of many other enzymes

9 K. Felix, K. Kelliher, G. W. Bornkamm, and S. Janz, Cancer Res. 58, 1616 (1998).

10 K. Felix, K. A. Kelliher, G. W. Bornkamm, and S. Janz, Cancer Res. 59, 3621 (1999).

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