The study of Gap Junctional Intercellular Communication in keratinocytes as screening of promoter effect induced by industrial and environmental toxic substances

Roberto Zefferino, G. Elia, Maria Lasalvia, Claudia Piccoli, D. Boffoli, N. Capitanio, L. Ambrosi

Research output: Contribution to journalArticle

Abstract

Background: Disordered functioning of gap junctions between normal and initiated cells has been proposed as one possible mechanism of tumour promotion. Many putative carcinogens such as peroxisome proliferators, are known to activate various signal transduction mechanisms and modulate gap junctional intercellular communication (GJIC). They act as tumour promoters on pre-existing "initiated" cells, rather than as genotoxic initiators. Objectives: The aim of this article is to provide a screening-tool to evaluate the promoter carcinogen effect of environmental and occupational chemical contaminants, focusing on their ability to alter GJIC. Methods: GJIC was investigated in serum-free cultured primary human keratinocytes, by directly evaluating the intercellular transfer of a microinjected fluorescent dye (Dye transfer). The expression of caspase 3, which is the ultimate target to be activated of both mitochondrial- and non-mitochondrial-linked pro-apoptotic pathways, was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Results: Mercury chloride (10 nM), mono-methyl Mercury (250 nM) and Trichloroethylene (500 μM) were shown to significantly inhibit GJIC. Conversely di-methyl mercury, lead acetate and epichloridine had no effect on GJIC. All Trans Retinoic Acid completely reversed the inhibitory effect on GJIC induced by HgCl 2, but not that induced by mono-methyl mercury and trichloroethylene. The result of a RTPCR assay on total RNA cell extract showed that treatment of keratinocytes with 10 nM HgCl 2 resulted in a decrease of the pro-apoptotic caspase 3 expression. Conclusions: In this work a protocol is designed to study gap junction intercellular communication in primary cultures of human keratinocytes which could be used as a reliable screening tool to test the promoter carcinogen effect of various environmental and occupational contaminants.

Original languageEnglish
Pages (from-to)222-230
Number of pages9
JournalMedicina del Lavoro
Volume96
Issue number3
Publication statusPublished - 2005

Fingerprint

Hazardous Substances
Mercury
Keratinocytes
Trichloroethylene
Gap Junctions
Caspase 3
Carcinogens
Carcinogenicity Tests
Environmental Carcinogens
Peroxisome Proliferators
Aptitude
Tretinoin
Cell Extracts
Reverse Transcriptase Polymerase Chain Reaction
Fluorescent Dyes
Chlorides
Signal Transduction
Coloring Agents
RNA
Serum

Keywords

  • Apoptosis
  • Cancer
  • Keratinocytes
  • Mercury
  • Trichloroethylene

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health

Cite this

The study of Gap Junctional Intercellular Communication in keratinocytes as screening of promoter effect induced by industrial and environmental toxic substances. / Zefferino, Roberto; Elia, G.; Lasalvia, Maria; Piccoli, Claudia; Boffoli, D.; Capitanio, N.; Ambrosi, L.

In: Medicina del Lavoro, Vol. 96, No. 3, 2005, p. 222-230.

Research output: Contribution to journalArticle

Zefferino, Roberto ; Elia, G. ; Lasalvia, Maria ; Piccoli, Claudia ; Boffoli, D. ; Capitanio, N. ; Ambrosi, L. / The study of Gap Junctional Intercellular Communication in keratinocytes as screening of promoter effect induced by industrial and environmental toxic substances. In: Medicina del Lavoro. 2005 ; Vol. 96, No. 3. pp. 222-230.
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AU - Zefferino, Roberto

AU - Elia, G.

AU - Lasalvia, Maria

AU - Piccoli, Claudia

AU - Boffoli, D.

AU - Capitanio, N.

AU - Ambrosi, L.

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AB - Background: Disordered functioning of gap junctions between normal and initiated cells has been proposed as one possible mechanism of tumour promotion. Many putative carcinogens such as peroxisome proliferators, are known to activate various signal transduction mechanisms and modulate gap junctional intercellular communication (GJIC). They act as tumour promoters on pre-existing "initiated" cells, rather than as genotoxic initiators. Objectives: The aim of this article is to provide a screening-tool to evaluate the promoter carcinogen effect of environmental and occupational chemical contaminants, focusing on their ability to alter GJIC. Methods: GJIC was investigated in serum-free cultured primary human keratinocytes, by directly evaluating the intercellular transfer of a microinjected fluorescent dye (Dye transfer). The expression of caspase 3, which is the ultimate target to be activated of both mitochondrial- and non-mitochondrial-linked pro-apoptotic pathways, was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Results: Mercury chloride (10 nM), mono-methyl Mercury (250 nM) and Trichloroethylene (500 μM) were shown to significantly inhibit GJIC. Conversely di-methyl mercury, lead acetate and epichloridine had no effect on GJIC. All Trans Retinoic Acid completely reversed the inhibitory effect on GJIC induced by HgCl 2, but not that induced by mono-methyl mercury and trichloroethylene. The result of a RTPCR assay on total RNA cell extract showed that treatment of keratinocytes with 10 nM HgCl 2 resulted in a decrease of the pro-apoptotic caspase 3 expression. Conclusions: In this work a protocol is designed to study gap junction intercellular communication in primary cultures of human keratinocytes which could be used as a reliable screening tool to test the promoter carcinogen effect of various environmental and occupational contaminants.

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