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Newsletter, February 14, 2001

Environmental Mutagen Society Special Interest Group in: Transgenic and In Vivo Mutagenesis

Standard Disclaimer :  This Newsletter does not necessarily reflect the opinions or positions of the EMS or its members. Any errors or omissions are strictly on the part of the secretary.

Impending EMS 2001 : 

            I've just been watching the Buick Invitational at Torrey Pines GC, near San Diego.  If you haven't visited Torrey Pines National Park (across the road from the GC), make it one of your stops for fresh air and a walk when at EMS 2001.  Beachcombing for neat shells is also fun there.  Torrey Pines Park is right next door to the Scripps, Stratagene, and near the shopping paradise of La Jolla. Lucky dogs.  Also in San Diego, if you have a free day, try to get over to the desert in Anza-Borrego (http://www.desertusa.com/anza_borrego/du-abpmain.html,  a couple of hours drive) to see the blooming desert.  Astounding.  March is supposed to be the peak flowering, but it sort of depends on the weather.  Stop in Julian for great apple pie on the way..

            Also when in San Diego, the TIVM group will be holding a breakfast meeting.  If you have data or some interesting results you would like to present for discussion, please forward a brief note to Carrie (cvalentine@nctr.fda.gov).  Ideally, 3-4 presentations, say 5-10 minutes to present (skip the preambles), and 5-10 to discuss, might be a good format.  Overheads, not video or slides. If you would like to distribute anything in advance of the meeting, send it to Carrie or Barry Ford for redistribution to the list.

Science Stuff :

The Winter 2001 EMS Newsletter (Committee Reports section) contains an article by our current Chair, Carrie Valentine, on the sensitivity of transgenic mutation assays.  Several of you have taken the time to comment on this article.  We’d like to circulate these comments before the San Diego meeting in order to stimulate discussion at our interest group meeting on Thursday morning.

John Heddle writes:

1.  A fold increase in mutation is not the proper way to look at the  data.  It is statistical power that matters and that depends both on the number of mutations detected and on the frequencies.  It would be, for example, far more reliable to detect at change in the number of mutants from 50 to 90, less than a doubling, than from 1 to 3, a tripling if all of the sample sizes were the same.  So the spontaneous frequency is not the only factor, quite aside from the fact that the hprt assay is a beast that no one would use as a test and is available in only one or two tissues at the moment and is much LESS sensitive that lacI except in very young animals.  But the statistics are the important point. 

Carrie:  Of course, John is right about statistical power.  Fold increase conveys the idea of a signal-to-noise ratio, but the significance of the increase is determined by the statistical analysis.  By the way, scoring for the hprt assay (or any assay detecting growth of colonies) has been automated using the viability indicator alamarBlue and a fluorescence plate reader and is reported in the current issue of Environ. Molec. Mutagen. (Dobrovolsky et al., 2000 36:283-91).

2.      Ex vivo mutations will not be a problem so long as people use an adequate manifestation time [Heddle, J.A., Mutant Manifestation:  The Time Factor in Somatic Mutagenesis.  Mutagenesis 14: 1-3 (1999).].  In our work in the intestine, for example, we always allow at least one week and usually two after the last treatment before making a measurement.  This means that the adducts have been hugely diluted and, probably, either repaired or fixed [Bielas, J., and Heddle, J.A., Proliferation is necessary for both repair and mutation in transgenic mouse cells.  Proc. Natl. Acad. Sci.  97:11391-6  (2000).]. 

Carrie:  This is true for ex vivo mutations resulting from adducts of the treatment mutagen.  In his very interesting PNAS article John showed that induced Big Blue mosaic plaques disappeared to the background level of 0.5% before two cell divisions in cell culture. 

George Douglas writes:

The fact that the transgenic model has a higher spontaneous mutant frequency should not a priori be an impediment to its use as a routine assay.  The hprt assay is limited to a very few tissues (principally spleen), and is, therefore, of limited value in permitting the study of mutations in the diversity of target tissues for cancer, or in germ cells.

The lacZ system with P-gal positive selection does not appear to yield any mosaic plaques.  This is presumably because any bacterium carrying a phage with an adduct would be killed as a wild type before the adduct could be converted to a mutation.  Yet, variation in the mutant frequency among tissues is still seen.  As for DNA extraction causing differential effects, we should not see it in the lacZ model.   SEE ALSO    Bielas JH, Heddle JA. Proliferation is necessary for both repair and mutation in transgenic mouse cells.  Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11391-6. 
[Commentary by Pat O’Neill, pp 11137-11139.]

Carrie:  George reported in last year’s EMS abstracts² that the spontaneous mutant frequencies for germ cells, bone marrow, and liver were 2.5, 5.2 and 6.9 [x 10^-5] respectively.   Is the 2.5 x 10^-5 spontaneous mutation frequency for the lacZ transgene determined by the mutation rate in E. coli of the selection system?

Tom Skopek writes:

One point for your consideration.  On p.36 (first column) you say that "solace is also gained from pointing out that the typical spontaneous mutant frequency for diploid genes of similar function (aprt and tk) are 10-fold higher than for hprt".  This is true for total mutants, but if you consider the frequency of non-recombination-derived mutants, the frequency is in the mid-10-6 range, ball park with hprt.  Mutants at hprt cannot be derived from recombination so comparing its frequency to total tk or aprt is an apple/orange situation.  Agree?   Do we then evoke that hprt, tk, and aprt are all selected against in vivo?

Your point that the low lacI frequency in rat bladder and bone marrow proves that "ex vivo" mutations are not driving the high mutation frequency seen in other tissues is a good one.  In addition we also argue that this lower frequency is probably closer to the "real" transgene spontaneous rate in vivo, given that one can artifactually raise mutation frequency through a variety of mechanisms, but it is difficult to conceive of a mechanism that would artifactually decrease frequency.  As you probably have heard from Manju, we have a paper in press in MR that shows that lacI is fully methylated in bone marrow, which kind of throws a monkey wrench into the whole CpG mechanism for spontaneous mutations.  Clearly, more research is necessary (job security for the likes of us).

Carrie: I do agree that the total mutant frequency of Hprt cannot be directly compared to Tk and Aprt; somehow I forgot that the spectra are different.  The similar parts of the mutational spectrum are what should be compared.   This leads us back to Barbara Parson’s thesis¹ that tissue specificity is what determines the background mutation frequency.  However, we still don’t have an explanation as to why the lacI and hprt gene have different spontaneous mutation frequencies in the same tissue, splenic lymphocytes.

¹McKinzie, PB, RR Delongchamp, RH Heflich, BL Parsons. 2001.  Propects for applying genotypic selection of somatic oncomutation to chemical risk assessment. Soon to be submitted to Mutation Research.

²Douglas, GR, Gingerich JD, Soper LM.  2000.  Lower spontaneous lacZ mutant frequency in Muta™Mouse male germ cells than in somatic tissues.  Environ. Mol. Mutagen. 35 Suppl. 31, p. 21.

Some Useful Information:
Internet Sites on TIVM (a sampling)

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All submissions / suggestions considered.  email to  barry.ford@drdc-rddc.gc.ca