Promotion of morphological transformation by Di-n-butyltin dichloride in C3H/10T1/2 cells: prediction by prior expression of tumour promoter-responsive genes

Our plain-English summary of Promotion of morphological transformation by Di-n-butyltin dichloride in C3H/10T1/2 cells: prediction by prior expression of tumour promoter-responsive genes, by C.L. Parfett, T. Marquardt, R. Pilon (Food and Chemical Toxicology, 2000).

The study

• Authors: C.L. Parfett, T. Marquardt, R. Pilon
• Journal: Food and Chemical Toxicology
• Published: 2000;38(4):339-49
• Cited by: 9 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Previous studies in our laboratory have shown that chemical treatments may induce increases in proliferin gene family mRNA accumulation in cultured murine embryonic cells. Proliferin inductions are highly correlated with subsequent promotional outcomes during two-stage focus-formation assays in C3H/10T1/2 cell cultures. In work reported here, the strong affiliation between these two responses was further validated after treating cells with di-n-butyltin dichloride which is a polyvinyl chloride (PVC) plastic additive that often contaminates food and water. Increased proliferin expression and promotion of morphological transformation occurred at similar concentrations. Promotion of transformation was detected at di-n-butyltin dichloride concentrations of 80 nM (24 ng/ml) and above, if added to initiated cultures before confluent monolayers had formed. Proliferin induction and morphological transformation were both reduced in confluent cultures treated with di-n-butyltin dichloride, as compared to subconfluent cultures. Proliferin expression measured in near-confluent cultures was induced up to 10-fold during the 36-hr period following di-n-butyltin dichloride exposure and was accompanied by increased accumulation of transcripts from many genes regulated by oxidative stresses, growth-inducing agents, and/or other promoting agents (asbestos, superoxide radicals ). Di-n-butyltin dichloride-induced mRNA species included members of the fos and jun proto-oncogene families, c-myc, egr1, ribonucleotide reductase (R2 subunit), odc, macrophage chemotactic protein/je, hsp70, metallothionine IIA, c-sod and mn-sod. The observed patterns of RNA accumulation suggested that a small subset of mRNA species, including proliferin, exhibit regulatory behaviour as a response to dissimilar agents or conditions that promote focus-formation in C3H/10T1/2 cultures. Plausible predictions of promotional effects in two-stage morphological transformation assays can be made from gene-expression responses to test agents.

In plain English

This Health Canada laboratory study tested whether di-n-butyltin dichloride, a PVC plastic additive, promotes cancer-like changes (morphological transformation) in C3H/10T1/2 mouse embryo cells. Asbestos was not tested; it appears only in comparisons with the authors' earlier work. Cells were first treated for 24 hours with the carcinogen 3-methylcholanthrene. The tin compound was added 1.5 to 8 days later, and transformed cell clusters (foci) were counted 6 weeks after the carcinogen was removed. Added at 300 nM after 2 days, it produced 16 of the most transformed (type III) foci in 19 dishes. Added 4 or 8 days later, there was no significant increase. A second experiment found increases at 80 and 240 nM but not 27 nM. Proliferin gene activity rose far less in crowded (confluent) cultures. At 900 nM, hsp70, c-jun and egr1 activity rose 50-fold or more within 10 hours. The authors concluded that changes in specific genes, especially the proliferin family, are good predictors of transformation in this cell line; they said jun-B and other stress-regulated genes needed more assessment. They also said two-stage studies in living animals were required.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory experiment in cultured mouse cells: two-stage (initiation then promotion) cell transformation assays, plus measurements of gene activity (RNA levels)
• Who was studied: C3H/10T1/2 mouse embryo cell line, grown in dishes (20 dishes per treatment group, 500 cells per dish in the transformation assays), at Health Canada, Ottawa
• Exposure: Initiation with 93 nM 3-methylcholanthrene for 24 hours. Di-n-butyltin dichloride (27 to 240 nM, or 300 nM) was added 1.5 to 8 days after the carcinogen was removed. All cultures were kept for 6 weeks after that removal. Gene-activity experiments used 300, 700 or 900 nM. Asbestos was not tested in this study and is referred to only from the authors' earlier work.
• Main results:
  • Experiment A: in cultures given the carcinogen and then 300 nM di-n-butyltin dichloride 2 days later, there were 16 type III foci in 19 dishes. Cultures given the carcinogen alone had 0 in 19 dishes, and cultures given di-n-butyltin dichloride alone had 1 in 20 dishes. The authors described this as a significant, 16-fold increase.
  • Experiment A: transformed foci were not significantly increased when di-n-butyltin dichloride was added 4 or 8 days after the carcinogen was removed
  • Experiment B (di-n-butyltin dichloride added 1.5 days after the carcinogen): focus formation increased at 80 and 240 nM but not at 27 nM
  • Proliferin RNA rose much more in less dense cultures. In those cultures, the added accumulation was about 500 times (700 nM) and 200 times (300 nM) the low levels in confluent cultures given serum only. In confluent cultures, totals were about 90 times (700 nM) and 10-fold (300 nM) their controls.
  • In near-confluent cultures, proliferin expression rose up to 10-fold during the 36 hours after exposure
  • At 900 nM, hsp70, c-jun and egr1 RNA rose 50-fold or more within the first 10 hours in each experiment. No increase in catalase (cat) RNA was detected.
• The authors' conclusion: The authors concluded that chemically induced changes in the expression of specific genes, especially the proliferin gene family, serve as good predictive indicators of transformation outcome in the C3H/10T1/2 cell line
• Limitations the authors state:
  • The highest concentration of di-n-butyltin dichloride that promoted transformation was not determined
  • The authors said a more thorough assessment is required of the link between promotion of transformation and jun-B or other stress-regulated RNA species. Wider studies across cell densities, toxic and non-toxic concentrations, and other substances were beyond the scope of this investigation.
  • The authors said two-stage bioassays in living animals are required to examine whether these compounds act as tumour promoters

Topics

Medical terms in this study, explained in our Lexicon: Oxidative Stress, Superoxide, Macrophage.

Related studies

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