[Dioxin-l] RE: References about threshold.

[david bell]

Hi Diane
I agree that you are absolutely correct to point out the differences between 
molecular, cellular, whole organism, etc responses.

However, I will query your comment on the Lucier paper (s). As far as I can 
tell from the papers I can find (and I put the abstracts at the bottom), 
Lucier took the dose response down to 1 ng dioxin/kg- at which point he can 
see a small induction (two fold of protein, 10 fold of RNA) of P450. 1 ng/kg 
is 100 000 fold above the EPA TDI limit, and 1000 fold above human doses. 
These papers don't tell us that the dose-response continues indefinitely, 
and they make no comment about one molecule per cell. I suspect you have 
better knowledge of this than I, as you are looking at responses which occur 
at lower levels.

I am surprised to see the statement about ensuring a detectable response in 
the dioxin system with a single molecule- but obviously, I would love to see 
your sources. Am I reading you wrongly ?

It occurs to me that you are making a conceptual description of the 
mechanism- 1 molecule of dioxin + 1 molecule of AhR + 2x hsp90, etc. In this 
context, I guess concentrations are to some extent irrelevant; but then you 
are not making predictions about what will happen at what dose with this 
model.

Once you look at the cell- as you say, with all its complexities and 
signal-coupling- then thresholds seem fairly well characterised.

cheers
david

>As Lucier showed, when
>you get one molecule binding to a single receptor, you can ensure a
>detectable response (in the absence of a non-competitive antagonist), in
>this case induction of P4501A mRNA. <snip> So at the molecular level, 
>threshold
>doesn't really exist.

TI:  DIOXIN-RESPONSIVE GENES - EXAMINATION OF DOSE-RESPONSE RELATIONSHIPS 
USING QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION
AU:  VANDENHEUVEL_JP, CLARK_GC, KOHN_MC, TRITSCHER_AM, GREENLEE_WF, 
LUCIER_GW, BELL_DA
JN:  CANCER RESEARCH, 1994, Vol.54, No.1, pp.62-68
AB:  The purpose of the present experiments was to examine dose- response 
relationships for induction of hepatic mRNA following a single 
administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The 
induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other 
''dioxin-responsive'' genes including UDP- glucuronosyltransferase 1, 
plasminogen activator inhibitor 2, and transforming growth factor a using a 
sensitive reverse transcriptase- polymerase chain reaction-based method. 
Sample-to-sample variability in amplification is a concern in using 
polymerase chain reaction to quantitate biological responses. However, in 
the present study recombinant RNA templates were synthesized to use as 
internal standards in both the reverse transcription and the polymerase 
chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive 
to TCDD treatment with increases observed at doses as low as 1 ng/kg body 
weight. The induction of CYP1A1 mRNA correlated highly (R2 > 0.90) with an 
increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated 
enzyme activity. However, induction of CYP1A1 mRNA levels was detected at 
lower TCDD doses than was ethoxy-resorufin-o- deethylase activity, 
reflecting the greater sensitivity of the reverse transcription-polymerase 
chain reaction approach to detect transcriptional activation of the CYP1A1 
gene. UDP- glucuronosyltransferase 1 mRNA was increased over control 
(5-fold) but required 1000-times higher TCDD doses (1 mug/kg) to result in a 
significant increase than did CYP1A1. Plasminogen activator inhibitor 2 and 
transforming growth factor alpha mRNA, both previously shown to be induced 
by TCDD in human keratinocytes, were not increased in rat liver. Hence, 
these studies reaffirm that TCDD acts through classical receptor mechanisms 
with gene-to-gene differences in responsiveness. The reverse 
transcription-polymerase chain reaction method developed to measure mRNA for 
dioxin-reponsive genes in rat liver will allow for measuring multigene and 
tissue responses to TCDD and other xenobiotics with high sensitivity, 
reproducibility, and adaptability and should increase our understanding of 
various dose-response relationships.


DIOXIN-RESPONSIVE GENES - EXAMINATION OF DOSE-RESPONSE RELATIONSHIPS USING 
QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION
AU:  VANDENHEUVEL_JP, CLARK_GC, KOHN_MC, TRITSCHER_AM, GREENLEE_WF, 
LUCIER_GW, BELL_DA
JN:  CANCER RESEARCH, 1994, Vol.54, No.1, pp.62-68
AB:  The purpose of the present experiments was to examine dose- response 
relationships for induction of hepatic mRNA following a single 
administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The 
induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other 
''dioxin-responsive'' genes including UDP- glucuronosyltransferase 1, 
plasminogen activator inhibitor 2, and transforming growth factor a using a 
sensitive reverse transcriptase- polymerase chain reaction-based method. 
Sample-to-sample variability in amplification is a concern in using 
polymerase chain reaction to quantitate biological responses. However, in 
the present study recombinant RNA templates were synthesized to use as 
internal standards in both the reverse transcription and the polymerase 
chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive 
to TCDD treatment with increases observed at doses as low as 1 ng/kg body 
weight. The induction of CYP1A1 mRNA correlated highly (R2 > 0.90) with an 
increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated 
enzyme activity. However, induction of CYP1A1 mRNA levels was detected at 
lower TCDD doses than was ethoxy-resorufin-o- deethylase activity, 
reflecting the greater sensitivity of the reverse transcription-polymerase 
chain reaction approach to detect transcriptional activation of the CYP1A1 
gene. UDP- glucuronosyltransferase 1 mRNA was increased over control 
(5-fold) but required 1000-times higher TCDD doses (1 mug/kg) to result in a 
significant increase than did CYP1A1. Plasminogen activator inhibitor 2 and 
transforming growth factor alpha mRNA, both previously shown to be induced 
by TCDD in human keratinocytes, were not increased in rat liver. Hence, 
these studies reaffirm that TCDD acts through classical receptor mechanisms 
with gene-to-gene differences in responsiveness. The reverse 
transcription-polymerase chain reaction method developed to measure mRNA for 
dioxin-reponsive genes in rat liver will allow for measuring multigene and 
tissue responses to TCDD and other xenobiotics with high sensitivity, 
reproducibility, and adaptability and should increase our understanding of 
various dose-response relationships.




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