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Re: FW: Food irradiation article
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For those of you following the food irradiation issue, attached is the =
Public Citizen translation of the German study. Thanks Ernie, for digging =
this up.
Jim Hardeman
Jim_Hardeman@mail.dnr.state.ga.us
>>> "Faillace, Ernie" <FaillaceE@ttnus.com> 3/13/2001 8:35:40 >>>
Jim,
Here is the translated article sent to me by the author of the Washington
Post piece. If you think the rest of RADSAFE might benefit, feel free to
post it. I had hoped for a simple link to a web site containing the =
article
text, but this is probably the next best thing. Note that the source of =
the
translation is Public Citizen, so there may be some "bias" in the
translation. I think Franz may provide a better perspective on the =
accuracy
of this translation if he could get access to the original work.
Ernesto Faillace, Eng.D, CHP
Nuclear Engineer/Health Physicist
Tetra Tech NUS
900 Trail Ridge Rd
Aiken, SC 29803
(803) 649-7963 x303
(803) 642-8454 (fax)
faillacee@ttnus.com
- -----Original Message-----
From: Robert L. Wolke [mailto:wolke+@pitt.edu]
Sent: Monday, March 12, 2001 8:49 PM
To: Faillace, Ernie
Subject: Re: Food irradiation article
Here is the article.
Report by the Bundesforschungsanstalt f=FCr Ern=E4hrung
[Federal Nutrition Research Institute]
BFE-R-99-01
Food Irradiation
Fifth German Conference
Editors:
M. Kn=F6rr, D.A.E. Ehlermann and H. Delinc=E9e
Conference on November 11 & 13, 1998
BFE, Karlsruhe
Bundesforschungsanstalt f=FCr Ern=E4hrung
Karlsruhe
1999
Translated from the German by Public Citizen, Washington, D.C.
February 2001
Genotoxicity of 2-dodecylcyclobutanone
Henry Delinc=E9e, Beatrice-Louise Pool-Zobel and Gerhard Rechkemmer
Institut f=FCr Ern=E4hrungsphysiologie der Bundesforschungsanstalt f=FCr =
Ern=E4hrung
[Institute for Nutritional Physiology of the Federal Nutrition Research
Institute]
Haid-und-Neu-Str. 9, D-76131 Karlsruhe
Summary
In the treatment of foods containing fat with ionizing radiation - for
example,
the irradiation of chicken or hamburger to kill pathogens such as =
Salmonella
spp. or E. coli O.157:H7 - a range of lipolytic digestion products are
generated, among them the group of 2-alkylcyclobutanones. These compounds
contain the same number (n) of carbon atoms as their precursor fatty =
acids,
whereby a hydrocarbon chain with n-4 carbon atoms is attached to ring
position
2 of the cyclobutanone. In this way, 2-dodecylcyclobutanone is generated
from
palmitic acid. Up to the present day, cyclobutanones have not been found =
in
non-irradiated foods. Therefore, it is important to examine the toxic or
genotoxic potential of cyclobutanones in the context of discussions about
the
safety of irradiated foods
In this study, in vivo experiments were conducted on rats, which received
two
different doses of 2-dodecylcyclobutanones by way of pharyngeal probe.
After
16 hours, colon cells were isolated from the rat and analyzed for DNA =
damage
by
means of the comet assay.
No cytotoxic effects were detected in the trypan blue vitality test. When
the
"% tail intensity" or the "tail moment" was used in the comet assay for
quantitative analysis, the values obtained with an experimental group that
received a low concentration of 2-dodecylbutanone (1.12 mg/kg body weight)
were
similar to those of the control group, which was administered 2% dimethyl
sulfoxide. Slight but significant DNA damage was observed in the
experimental
group that received the higher concentration of 2-dodecylcyclobutanone =
(14.9
mg/kg body weight). Further studies are needed to clarify the relevance =
of
these results to an evaluation of risk from the consumption of irradiated
foods.
Introduction
Of late there has been growing interest in the treatment of foods with
ionizing
radiation. The irradiation can help improve the hygienic quality of the
food
and prevent diseases that otherwise could be caused by consumption of =
foods
contaminated with parasites or pathogenic microorganisms. Furthermore, =
the
irradiation of certain foods facilitates an improvement in the storage =
life
and
reduces the spoilage rate [Diehl, 1995]. A growing number of countries =
have
approved the use of ionizing radiation for numerous products [Anon., =
1998].
Within the EU, one can expect harmonization of the legal regulations of =
the
member states with regard to foods and food components treated with =
ionizing
radiation. As a first step, irradiation of dried aromatic herbs and =
spices
is
to be permitted in all EU nations. This development is based in part on =
the
positive evaluation of the procedure by the World Health Organization. In =
a
1992 position statement, WHO stated that "foods that have been treated =
with
ionizing radiation and produced according to good manufacturing practice
(GMP)
are to be regarded as safe in terms of health and satisfactory from the
perspective of nutritional physiology." Numerous studies and animal =
feeding
experiments, as well as experiments on volunteer test subjects, support =
this
conclusion [WHO, 1994]. Taking account of the studies available to date, =
a
new
expert committee concluded in 1997 that "even irradiation of foods with =
high
doses (> 10 kGy) may be judged safe and satisfactory in terms of nutrition"=
[WHO, 1997, 1998]. In recent years, there has also been increasing =
interest
in
analytical techniques to determine whether a product has been irradiated
[Delinc=E9e, 1998]. For example, a research team in Northern Ireland has
determined that certain lipolytic digestion products - namely, the
2-alkylcyclobutanones [LeTellier and Nawar, 1972] - might be products that
are
unique to irradiation and therefore hold great promise as markers of
irradiation treatment [Stevenson et al., 1990, Stevenson, 1996]. As a
result
of irradiation, the acyl-oxygen bond in triglycerides is cleaved, with
formation of 2-alkylcyclobutanones with the same number of carbon atoms as
the
initial fatty acid and with the alkyl group in ring position 2. For
example,
2-dodecylcyclobutanone and 2-tetradecylcyclobutanone are formed from
palmitic
acid and stearic acid, respectively. Although 2-methylcyclobutanone has
been
identified following ultrasound treatment of Hevea brasiliensis latex, for
example [Nishimura et al., 1977], cyclobutanones have not yet been =
detected
in
non-irradiated foods [Stevenson, 1996]. However, since cyclobutanones do
occur
in irradiated foods - for example, at levels of 0.3-0.6 =B5g
2-dodecylcyclobutanone/g fat/kGy in chickens [Stevenson et al., 1990, =
1993;
Boyd et al., 1991; Crone et al., 1992 a, b, 1993; Stevenson, 1996] - it is
necessary to characterize their potentially toxic features and undertake a
risk
evaluation.
In this study, the so-called "comet assay," a new test procedure that
detects
DNA damage in individual cells by means of microgel electrophoresis, has
been
employed as the toxicological test procedure [McKelvey-Martin et al., =
1993;
Fairbairn et al., 1995]. Rat colon cells , tissue in which tumors can be
generated under certain nutritional conditions, were used as the target
cells.
Materials and Methods
Materials
The test substance, 2-dodecylcyclobutanone (2-DCB) was synthesized =
according
to
the specifications of Boyd et al. (1991).
In vivo experiment
Male Sprague-Dawley rats (=BB 250 g) were obtained from Charles River Wiga
GmbH
(D-97633 Sulzfeld) and kept under the usual conditions. The rats were
randomly
divided into 4 groups. Two groups of six animals each received 2-DCB via
pharyngeal probe: the first group received 1.12 mg/kg body weight (BW), =
the
second group 14.9 mg/kg BW. A group of three animals served as negative
control, and received the solvent of 2-DCB, namely 2% dimethyl sulfoxide
(DMSO)
in physiological sodium chloride solution (5 ml/kg BW). The fourth group
with
three animals was employed as positive control, and received 15 mg
1,2-dimethylhydrazine (DMH)/kg BW (dissolved in physiological sodium
chloride
solution, 5 ml/kg BW). The feeding and treatment regimen employed here =
has
been described (Pool-Zobel et al., 1996). After 16 hours of exposure -
which
was determined to be the optimal period of time for the formation of DNA
damage
in colon cells caused by DMH and measurable by the comet assay [Pool-Zobel,=
1996] - the colon was removed from the rats and the colon cells isolated =
by
means of enzymatic digestion [Brendler-Schwaab et al., 1994].
Cyotoxicity
The potential cytotoxicity of 2-DCB to the cells of the colon was checked
with
the aid of the trypan blue vitality test, a rapid and simple method to
differentiate between living and non-living cells [Pool et al., 1990;
Pool-Zobel et al., 1994].
Comet assay
DNA damage to the colon cells was determined by means of single-cell
microgel
electrophoresis (comet assay) [Pool-Zobel et al., 1994; Pool-Zobel and
Leucht,
1997]. For each data point, 50 cells per slide and 3 slides per
determination
were analyzed. The evaluation was carried out on a fluorescence microscope=
with the image processing system of Perceptive Instruments (Halstead, =
Great
Britain). The DNA distribution in the comet was calculated as "% tail
intensity" and "tail moment" - the latter a product of the proportion of =
DNA
in
the tail and the length of the comet tail [Fairbairn et al., 1995]. With
more
severe damage to the DNA, the proportion of DNA in the tail, and hence =
also
the
"% tail intensity" and "tail moment," increase.
Determination of the quantity of substance administered
Two different concentrations of 2-DCB were selected. The low concentration=
was
meant to model radiation pasteurization (e.g. with 3 kGy), while the =
higher
concentration was intended to represent radiation sterilization (60 kGy).
For the radiation pasteurization (3 kGy) of fresh chicken, we assumed
formation
of =BB1.5 =B5g of 2-DCB/g fat. Since palmitic acid represents only about =
1/5 of
the fatty acids in chicken, the total quantity of cyclobutanones was =
roughly
projected to be 5 times as great. If one assumes at the same time that =
all
of
the fat that a person consumes is irradiated (according to the DGE
- -Nutrition
Report 1996, a man weighing 70 kg consumes an average of 104 g fat/day, or
1.49
g fat/kg BW), this would lead to a 2-DCB content of 1.5 =B5g x 5 x 1.49 =
=3D 11.2
=B5g
of 2-DCB/kg BW.
With a safety factor [Classen et al., 1987] of 10 for individual
differences,
and an additional factor of 10 to account for differences between various
species (here, rat/human), the expected no-effect level (NOEL) for =
radiation
pasteurization lies at
11.2 =B5g x 10 x 10 =3D 1.12 mg 2-DCB/kg BW.
Similarly, one would expect a NOEL of
20 =B5g x 5 x 1.49 x 10 x 10 =3D 1.49 mg 2-DCB/kg BW
for the radiation sterilization (60 kGy) of frozen chicken. This
calculation
is based on formation of =BB20 =B5g of 2-DCB/g fat for radiation sterilized=
(60
kGy), frozen (-46=B0 C) chicken [Crone et al., 1992a].
Results
The trypan blue vitality test did not reveal any cytotoxic effects on the
colon
cells from the 2-DCB that was administered. The vitality of the treated
cells
was on the same order of magnitude (=BB90%) as the cells of the negative
control
group, which were treated with DMSO alone.
On the other hand, DNA damage from 2-DCB was observed in the comet assay.
In
the evaluation of the comets, both as "% tail intensity" and as "tail
moment,"
the DNA damage exceeded that found in the negative control group. In the
group
of six animals that received the lower concentration of 1.12 mg 2-DCB/kg =
BW,
two of the animals exhibited increased DNA damage, while four of the =
animals
exhibited values like those of the control group (Fig. 1a).
When the results of the experimental group animals were combined, there =
was
no
significant difference relative to the negative control group (Fig. 1b). =
At
the higher concentration of 14.9 mg 2-DCB/kg BW, an increased level of DNA
damage was also detectable in the group, relative to the negative control
group
(Fig. 1b). While the increase in DNA damage is slight compared to the
positive
control group, which received DMN as alkylating agent, one must recall =
that
the
latter is a strong and specific rat colon carcinogen.
Fig. 1a Effect on Individual Animals
Fig. 1b Effect on Groups of Animals
Figures 1a, b DNA single-strand breaks in rat colon cells from the action =
of
2-dodecylcyclobutanone and DMSO, or DMH. Administered with pharyngeal =
probe
16
hours before isolation of the colon.
(** p < 0.01 significantly different from the negative control with =
DMSO;
unpaired, two-sided Student's t-test, n =3D 3-6).
Discussion
Initial in vitro experiments with 2-dodecylcyclobutanone, which at various
concentrations was applied to rat colon cells as well as colon cells from
human
biopsies, have shown that 2-DCB leads to DNA damage [Delinc=E9e and
Pool-Zobel,
1998]. Although the concentrations of 2-DCB that were used, ranging from
0.30
- - 1.25 mg/ml, are large in comparison to the expected consumption of =B5g
quantities of 2-DCB, further clarification is needed to determine whether
the
these results are relevant to the safety of irradiated foods.
The in vivo experiments that were just conducted likewise show DNA damage =
to
colon cells at higher concentrations of 2-DCB. Of course, one must keep =
in
mind that not every instance of DNA damage proves to be a precursor to
damage
severe enough to generate a tumor, or leads to mutations in tumor-relevant
genes. Furthermore, possible DNA repair processes and other cytotoxic
events,
for instance apoptosis, play a role before lesions become manifest and =
cell
degeneration is initiated.
In addition, the quantity of 2-DCB that was administered here is to be
regarded
as very high. A projection shows that the concentration of 14.9 mg/kg BW =
in
humans corresponds to consumption of more than 800 radiation-sterilized =
(60
kGy) broiler chickens. This comparison raises the question of whether the
safety factors must in fact be 10 x 10. With several food ingredients =
(e.g.
selenium), this concept would lead to deficiency symptoms, since the =
amount
required in rats, for example, is about 25% of the toxic dose [Classen et
al.,
1987]. With lower safety factors, and hence lower test concentrations of
2-DCB, there would no longer be any detectable DNA damage.
It should be mentioned once again that in many animal feeding experiments
with
irradiated foods in which it is known that cyclobutanone was also in the
feed,
no evidence has been found to indicate an injury from irradiated foods =
that
have been consumed. Typical in this regard is the Raltech study in the =
USA
[Thayer et al., 1987], in which several generations of mice and dogs were
fed
with radiation-sterilized chicken. This study also included
nutrition-physiological, teratological and genotoxic experiments on =
various
species of animal.
In each case, it is necessary to check the relevance of the results that
have
been obtained. It is striking that the variation in observations is much
greater at the low dose than the high dose, which in the latter case =
entails
statistical significance. This must also be clarified.
Conclusion
High concentrations of 2-dodecylcyclobutanone lead to DNA damage in colon
cells
that is detectable with the comet assay. The requisite concentrations are
very
much higher than those that can be reached through the consumption of
irradiated foods that contain fat. The results urge caution, and should
provide impetus for further studies.
Note of thanks
We thank Ms. R. Lambertz and Mr. M. Knoll for their excellent technical
assistance. We also thank Dr. C. H. McMurray (The Department of Agricultur=
e
for Northern Ireland, Belfast, United Kingdom) for providing the
2-dodecylcyclobutanone, and the International Consultative Group on Food
Irradiation (ICGFI) for partial financial support.
Bibliography
[only German references given here]
Classen, H. G., P. S. Elias and W. P. Hammes (1987). Toxicological-hygieni=
c
evaluation of food ingredients and additives as well as serious
contaminants.
Paul Parey, Berlin and Hamburg.
DGE (1996). Nutritional Status of the Federal Republic of Germany.
Nutrition
Report 1996, DGE, Frankfurt am Main, p. 43.
"Faillace, Ernie" wrote:
> Dr. Wolke,
>
> Could you please indicate a web link to the English translation of the
1998
> German study referenced in your March 7 Washington Post article (Nuclear
> Reactions)? Otherwise, could you please forward or attach the article =
in
> reply to this message?
>
> Thank you.
>
> Ernesto Faillace, Eng.D, CHP
> Nuclear Engineer/Health Physicist
> Tetra Tech NUS
> 900 Trail Ridge Rd
> Aiken, SC 29803
> (803) 649-7963 x303
> (803) 642-8454 (fax)
> faillacee@ttnus.com
- --
Robert L. Wolke
http://www.professorscience.com
- --=_134894D5.6A0B6B42
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<DIV><FONT size=3D1></FONT>For those of you following the food irradiation =
issue,=20
attached is the Public Citizen translation of the German study. Thanks =
Ernie,=20
for digging this up.</DIV>
<DIV> </DIV>
<DIV>Jim Hardeman</DIV>
<DIV>Jim_Hardeman@mail.dnr.state.ga.us<BR><BR>>>> "Faillace, =
Ernie"=20
<FaillaceE@ttnus.com> 3/13/2001 8:35:40 >>><BR>Jim,<BR><BR>H=
ere=20
is the translated article sent to me by the author of the Washington<BR>Pos=
t=20
piece. If you think the rest of RADSAFE might benefit, feel free=20
to<BR>post it. I had hoped for a simple link to a web site containing=
the=20
article<BR>text, but this is probably the next best thing. Note that =
the=20
source of the<BR>translation is Public Citizen, so there may be some =
"bias" in=20
the<BR>translation. I think Franz may provide a better perspective =
on the=20
accuracy<BR>of this translation if he could get access to the original=20
work.<BR><BR>Ernesto Faillace, Eng.D, CHP<BR>Nuclear Engineer/Health=20
Physicist<BR>Tetra Tech NUS<BR>900 Trail Ridge Rd<BR>Aiken, SC 29803<BR>(80=
3)=20
649-7963 x303<BR>(803) 642-8454=20
(fax)<BR>faillacee@ttnus.com<BR><BR><BR>-----Original Message-----<BR>From:=
=20
Robert L. Wolke [<A=20
href=3D"mailto:wolke+@pitt.edu]";>mailto:wolke+@pitt.edu]</A><BR>Sent: =
Monday,=20
March 12, 2001 8:49 PM<BR>To: Faillace, Ernie<BR>Subject: Re: Food =
irradiation=20
article<BR><BR><BR>Here is the article.<BR><BR>Report by the=20
Bundesforschungsanstalt f=FCr Ern=E4hrung<BR>[Federal Nutrition Research=20=
Institute]<BR>BFE-R-99-01<BR><BR><BR><BR><BR>Food Irradiation<BR>Fifth =
German=20
Conference<BR><BR><BR><BR><BR>Editors:<BR>M. Kn=F6rr, D.A.E. Ehlermann and =
H.=20
Delinc=E9e<BR><BR><BR><BR><BR>Conference on November 11 & 13, =
1998<BR>BFE,=20
Karlsruhe<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR>Bundesforschun=
gsanstalt=20
f=FCr Ern=E4hrung<BR>Karlsruhe<BR><BR>1999<BR><BR><BR><BR>Translated from =
the German=20
by Public Citizen, Washington, D.C.<BR>February 2001<BR><BR>Genotoxicity =
of=20
2-dodecylcyclobutanone<BR><BR>Henry Delinc=E9e, Beatrice-Louise Pool-Zobel =
and=20
Gerhard Rechkemmer<BR><BR>Institut f=FCr Ern=E4hrungsphysiologie der=20
Bundesforschungsanstalt f=FCr Ern=E4hrung<BR>[Institute for Nutritional =
Physiology=20
of the Federal Nutrition Research<BR>Institute]<BR>Haid-und-Neu-Str. 9, =
D-76131=20
Karlsruhe<BR><BR><BR>Summary<BR>In the treatment of foods containing fat =
with=20
ionizing radiation - for<BR>example,<BR>the irradiation of chicken or =
hamburger=20
to kill pathogens such as Salmonella<BR>spp. or E. coli O.157:H7 - a range =
of=20
lipolytic digestion products are<BR>generated, among them the group of=20
2-alkylcyclobutanones. These compounds<BR>contain the same number =
(n) of=20
carbon atoms as their precursor fatty acids,<BR>whereby a hydrocarbon =
chain with=20
n-4 carbon atoms is attached to ring<BR>position<BR>2 of the=20
cyclobutanone. In this way, 2-dodecylcyclobutanone is=20
generated<BR>from<BR>palmitic acid. Up to the present day, cyclobutan=
ones=20
have not been found in<BR>non-irradiated foods. Therefore, it is =
important=20
to examine the toxic or<BR>genotoxic potential of cyclobutanones in the =
context=20
of discussions about<BR>the<BR>safety of irradiated foods<BR><BR>In this =
study,=20
in vivo experiments were conducted on rats, which received<BR>two<BR>differ=
ent=20
doses of 2-dodecylcyclobutanones by way of pharyngeal probe.<BR>After<BR>16=
=20
hours, colon cells were isolated from the rat and analyzed for DNA=20
damage<BR>by<BR>means of the comet assay.<BR><BR>No cytotoxic effects =
were=20
detected in the trypan blue vitality test. When<BR>the<BR>"% tail=20
intensity" or the "tail moment" was used in the comet assay for<BR>quantita=
tive=20
analysis, the values obtained with an experimental group that<BR>received =
a low=20
concentration of 2-dodecylbutanone (1.12 mg/kg body weight)<BR>were<BR>simi=
lar=20
to those of the control group, which was administered 2%=20
dimethyl<BR>sulfoxide. Slight but significant DNA damage was =
observed in=20
the<BR>experimental<BR>group that received the higher concentration of=20
2-dodecylcyclobutanone (14.9<BR>mg/kg body weight). Further studies =
are=20
needed to clarify the relevance of<BR>these results to an evaluation of =
risk=20
from the consumption of irradiated<BR>foods.<BR><BR>Introduction<BR>Of =
late=20
there has been growing interest in the treatment of foods=20
with<BR>ionizing<BR>radiation. The irradiation can help improve =
the=20
hygienic quality of the<BR>food<BR>and prevent diseases that otherwise =
could be=20
caused by consumption of foods<BR>contaminated with parasites or pathogenic=
=20
microorganisms. Furthermore, the<BR>irradiation of certain foods=20
facilitates an improvement in the storage life<BR>and<BR>reduces the =
spoilage=20
rate [Diehl, 1995]. A growing number of countries have<BR>approved =
the use=20
of ionizing radiation for numerous products [Anon., 1998].<BR>Within the =
EU, one=20
can expect harmonization of the legal regulations of the<BR>member states =
with=20
regard to foods and food components treated with ionizing<BR>radiation.&nbs=
p; As=20
a first step, irradiation of dried aromatic herbs and spices<BR>is<BR>to =
be=20
permitted in all EU nations. This development is based in part on=20
the<BR>positive evaluation of the procedure by the World Health=20
Organization. In a<BR>1992 position statement, WHO stated that =
"foods that=20
have been treated with<BR>ionizing radiation and produced according to =
good=20
manufacturing practice<BR>(GMP)<BR>are to be regarded as safe in terms of =
health=20
and satisfactory from the<BR>perspective of nutritional physiology." =
=20
Numerous studies and animal feeding<BR>experiments, as well as experiments =
on=20
volunteer test subjects, support this<BR>conclusion [WHO, 1994]. =
Taking=20
account of the studies available to date, a<BR>new<BR>expert committee =
concluded=20
in 1997 that "even irradiation of foods with high<BR>doses (> 10 kGy) =
may be=20
judged safe and satisfactory in terms of nutrition"<BR>[WHO, 1997, =
1998]. =20
In recent years, there has also been increasing interest<BR>in<BR>analytica=
l=20
techniques to determine whether a product has been irradiated<BR>[Delinc=E9=
e,=20
1998]. For example, a research team in Northern Ireland has<BR>determ=
ined=20
that certain lipolytic digestion products - namely, the<BR>2-alkylcyclobuta=
nones=20
[LeTellier and Nawar, 1972] - might be products that<BR>are<BR>unique =
to=20
irradiation and therefore hold great promise as markers of<BR>irradiation=
=20
treatment [Stevenson et al., 1990, Stevenson, 1996]. As a<BR>result<B=
R>of=20
irradiation, the acyl-oxygen bond in triglycerides is cleaved, with<BR>form=
ation=20
of 2-alkylcyclobutanones with the same number of carbon atoms=20
as<BR>the<BR>initial fatty acid and with the alkyl group in ring =
position=20
2. For<BR>example,<BR>2-dodecylcyclobutanone and 2-tetradecylcyclobut=
anone=20
are formed from<BR>palmitic<BR>acid and stearic acid, respectively. =
=20
Although 2-methylcyclobutanone has<BR>been<BR>identified following =
ultrasound=20
treatment of Hevea brasiliensis latex, for<BR>example [Nishimura et al., =
1977],=20
cyclobutanones have not yet been detected<BR>in<BR>non-irradiated foods=20
[Stevenson, 1996]. However, since cyclobutanones do<BR>occur<BR>in=20=
irradiated foods - for example, at levels of 0.3-0.6=20
=B5g<BR>2-dodecylcyclobutanone/g fat/kGy in chickens [Stevenson et al., =
1990,=20
1993;<BR>Boyd et al., 1991; Crone et al., 1992 a, b, 1993; Stevenson, =
1996] - it=20
is<BR>necessary to characterize their potentially toxic features and =
undertake=20
a<BR>risk<BR>evaluation.<BR><BR>In this study, the so-called "comet =
assay," a=20
new test procedure that<BR>detects<BR>DNA damage in individual cells by =
means of=20
microgel electrophoresis, has<BR>been<BR>employed as the toxicological =
test=20
procedure [McKelvey-Martin et al., 1993;<BR>Fairbairn et al., 1995]. =
Rat=20
colon cells , tissue in which tumors can be<BR>generated under certain=20
nutritional conditions, were used as the target<BR>cells.<BR><BR>Materials =
and=20
Methods<BR>Materials<BR>The test substance, 2-dodecylcyclobutanone (2-DCB) =
was=20
synthesized according<BR>to<BR>the specifications of Boyd et al.=20
(1991).<BR><BR>In vivo experiment<BR>Male Sprague-Dawley rats (=BB 250 g) =
were=20
obtained from Charles River Wiga<BR>GmbH<BR>(D-97633 Sulzfeld) and kept =
under=20
the usual conditions. The rats were<BR>randomly<BR>divided into 4=20
groups. Two groups of six animals each received 2-DCB via<BR>pharynge=
al=20
probe: the first group received 1.12 mg/kg body weight (BW), the<BR>second =
group=20
14.9 mg/kg BW. A group of three animals served as negative<BR>control=
, and=20
received the solvent of 2-DCB, namely 2% dimethyl sulfoxide<BR>(DMSO)<BR>in=
=20
physiological sodium chloride solution (5 ml/kg BW). The fourth=20
group<BR>with<BR>three animals was employed as positive control, and =
received 15=20
mg<BR>1,2-dimethylhydrazine (DMH)/kg BW (dissolved in physiological=20
sodium<BR>chloride<BR>solution, 5 ml/kg BW). The feeding and =
treatment=20
regimen employed here has<BR>been described (Pool-Zobel et al., 1996). =
;=20
After 16 hours of exposure -<BR>which<BR>was determined to be the optimal =
period=20
of time for the formation of DNA<BR>damage<BR>in colon cells caused by DMH =
and=20
measurable by the comet assay [Pool-Zobel,<BR>1996] - the colon was =
removed from=20
the rats and the colon cells isolated by<BR>means of enzymatic digestion=20=
[Brendler-Schwaab et al., 1994].<BR><BR>Cyotoxicity<BR>The potential=20
cytotoxicity of 2-DCB to the cells of the colon was checked<BR>with<BR>the =
aid=20
of the trypan blue vitality test, a rapid and simple method to<BR>different=
iate=20
between living and non-living cells [Pool et al., 1990;<BR>Pool-Zobel et =
al.,=20
1994].<BR><BR>Comet assay<BR>DNA damage to the colon cells was determined =
by=20
means of single-cell<BR>microgel<BR>electrophoresis (comet assay) =
[Pool-Zobel et=20
al., 1994; Pool-Zobel and<BR>Leucht,<BR>1997]. For each data point, =
50=20
cells per slide and 3 slides per<BR>determination<BR>were analyzed. =
The=20
evaluation was carried out on a fluorescence microscope<BR>with the =
image=20
processing system of Perceptive Instruments (Halstead, Great<BR>Britain).&n=
bsp;=20
The DNA distribution in the comet was calculated as "% tail<BR>intensity" =
and=20
"tail moment" - the latter a product of the proportion of DNA<BR>in<BR>the =
tail=20
and the length of the comet tail [Fairbairn et al., 1995]. =20
With<BR>more<BR>severe damage to the DNA, the proportion of DNA in the =
tail, and=20
hence also<BR>the<BR>"% tail intensity" and "tail moment,"=20
increase.<BR><BR>Determination of the quantity of substance administered<BR=
>Two=20
different concentrations of 2-DCB were selected. The low=20
concentration<BR>was<BR>meant to model radiation pasteurization (e.g. with =
3=20
kGy), while the higher<BR>concentration was intended to represent =
radiation=20
sterilization (60 kGy).<BR><BR>For the radiation pasteurization (3 kGy) of =
fresh=20
chicken, we assumed<BR>formation<BR>of =BB1.5 =B5g of 2-DCB/g fat. =
Since=20
palmitic acid represents only about 1/5 of<BR>the fatty acids in chicken, =
the=20
total quantity of cyclobutanones was roughly<BR>projected to be 5 times =
as=20
great. If one assumes at the same time that all<BR>of<BR>the fat =
that a=20
person consumes is irradiated (according to the DGE<BR>-Nutrition<BR>Report=
=20
1996, a man weighing 70 kg consumes an average of 104 g fat/day, or<BR>1.49=
<BR>g=20
fat/kg BW), this would lead to a 2-DCB content of 1.5 =B5g x 5 x 1.49 =
=3D=20
11.2<BR>=B5g<BR>of 2-DCB/kg BW.<BR><BR>With a safety factor [Classen et =
al., 1987]=20
of 10 for individual<BR>differences,<BR>and an additional factor of 10 =
to=20
account for differences between various<BR>species (here, rat/human), =
the=20
expected no-effect level (NOEL) for radiation<BR>pasteurization lies=20
at<BR><BR>11.2 =B5g x 10 x 10 =3D 1.12 mg 2-DCB/kg BW.<BR><BR>Similarly, =
one would=20
expect a NOEL of<BR><BR>20 =B5g x 5 x 1.49 x 10 x 10 =3D 1.49 mg =
2-DCB/kg=20
BW<BR><BR>for the radiation sterilization (60 kGy) of frozen chicken. =
=20
This<BR>calculation<BR>is based on formation of =BB20 =B5g of 2-DCB/g fat =
for=20
radiation sterilized (60<BR>kGy), frozen (-46=B0 C) chicken [Crone et =
al.,=20
1992a].<BR><BR>Results<BR>The trypan blue vitality test did not reveal =
any=20
cytotoxic effects on the<BR>colon<BR>cells from the 2-DCB that was=20
administered. The vitality of the treated<BR>cells<BR>was on the =
same=20
order of magnitude (=BB90%) as the cells of the negative<BR>control<BR>grou=
p,=20
which were treated with DMSO alone.<BR><BR>On the other hand, DNA damage =
from=20
2-DCB was observed in the comet assay.<BR>In<BR>the evaluation of the =
comets,=20
both as "% tail intensity" and as "tail<BR>moment,"<BR>the DNA damage =
exceeded=20
that found in the negative control group. In the<BR>group<BR>of =
six=20
animals that received the lower concentration of 1.12 mg 2-DCB/kg =
BW,<BR>two of=20
the animals exhibited increased DNA damage, while four of the=20
animals<BR>exhibited values like those of the control group (Fig.=20
1a).<BR><BR>When the results of the experimental group animals were =
combined,=20
there was<BR>no<BR>significant difference relative to the negative control =
group=20
(Fig. 1b). At<BR>the higher concentration of 14.9 mg 2-DCB/kg BW, =
an=20
increased level of DNA<BR>damage was also detectable in the group, =
relative to=20
the negative control<BR>group<BR>(Fig. 1b). While the increase in =
DNA=20
damage is slight compared to the<BR>positive<BR>control group, which =
received=20
DMN as alkylating agent, one must recall that<BR>the<BR>latter is a strong =
and=20
specific rat colon carcinogen.<BR>Fig. 1a Effect on Individual=20
Animals<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR>=
<BR><BR><BR>Fig.=20
1b Effect on Groups of=20
Animals<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR>=
<BR><BR><BR><BR>Figures=20
1a, b DNA single-strand breaks in rat colon cells from the action=20
of<BR>2-dodecylcyclobutanone and DMSO, or DMH. Administered with=20
pharyngeal probe<BR>16<BR>hours before isolation of the colon.<BR>(** =
p=20
< 0.01 significantly different from the negative control with=20
DMSO;<BR>unpaired, two-sided Student's t-test, n =3D=20
3-6).<BR><BR>Discussion<BR>Initial in vitro experiments with=20
2-dodecylcyclobutanone, which at various<BR>concentrations was applied to =
rat=20
colon cells as well as colon cells from<BR>human<BR>biopsies, have shown =
that=20
2-DCB leads to DNA damage [Delinc=E9e and<BR>Pool-Zobel,<BR>1998]. =
Although=20
the concentrations of 2-DCB that were used, ranging from<BR>0.30<BR>- =
1.25=20
mg/ml, are large in comparison to the expected consumption of =B5g<BR>quant=
ities=20
of 2-DCB, further clarification is needed to determine whether<BR>the<BR>th=
ese=20
results are relevant to the safety of irradiated foods.<BR><BR>The in =
vivo=20
experiments that were just conducted likewise show DNA damage to<BR>colon =
cells=20
at higher concentrations of 2-DCB. Of course, one must keep =
in<BR>mind=20
that not every instance of DNA damage proves to be a precursor=20
to<BR>damage<BR>severe enough to generate a tumor, or leads to mutations =
in=20
tumor-relevant<BR>genes. Furthermore, possible DNA repair processes =
and=20
other cytotoxic<BR>events,<BR>for instance apoptosis, play a role before =
lesions=20
become manifest and cell<BR>degeneration is initiated.<BR><BR>In addition, =
the=20
quantity of 2-DCB that was administered here is to be<BR>regarded<BR>as =
very=20
high. A projection shows that the concentration of 14.9 mg/kg BW=20
in<BR>humans corresponds to consumption of more than 800 radiation-steriliz=
ed=20
(60<BR>kGy) broiler chickens. This comparison raises the question =
of=20
whether the<BR>safety factors must in fact be 10 x 10. With several =
food=20
ingredients (e.g.<BR>selenium), this concept would lead to deficiency =
symptoms,=20
since the amount<BR>required in rats, for example, is about 25% of the =
toxic=20
dose [Classen et<BR>al.,<BR>1987]. With lower safety factors, and =
hence=20
lower test concentrations of<BR>2-DCB, there would no longer be any =
detectable=20
DNA damage.<BR><BR>It should be mentioned once again that in many animal =
feeding=20
experiments<BR>with<BR>irradiated foods in which it is known that =
cyclobutanone=20
was also in the<BR>feed,<BR>no evidence has been found to indicate an =
injury=20
from irradiated foods that<BR>have been consumed. Typical in this =
regard=20
is the Raltech study in the USA<BR>[Thayer et al., 1987], in which =
several=20
generations of mice and dogs were<BR>fed<BR>with radiation-sterilized=20
chicken. This study also included<BR>nutrition-physiological,=20
teratological and genotoxic experiments on various<BR>species of=20
animal.<BR><BR>In each case, it is necessary to check the relevance of =
the=20
results that<BR>have<BR>been obtained. It is striking that the =
variation=20
in observations is much<BR>greater at the low dose than the high dose, =
which in=20
the latter case entails<BR>statistical significance. This must also =
be=20
clarified.<BR><BR>Conclusion<BR>High concentrations of 2-dodecylcyclobutano=
ne=20
lead to DNA damage in colon<BR>cells<BR>that is detectable with the =
comet=20
assay. The requisite concentrations are<BR>very<BR>much higher than =
those=20
that can be reached through the consumption of<BR>irradiated foods that =
contain=20
fat. The results urge caution, and should<BR>provide impetus for =
further=20
studies.<BR><BR>Note of thanks<BR>We thank Ms. R. Lambertz and Mr. M. =
Knoll for=20
their excellent technical<BR>assistance. We also thank Dr. C. H. =
McMurray=20
(The Department of Agriculture<BR>for Northern Ireland, Belfast, United =
Kingdom)=20
for providing the<BR>2-dodecylcyclobutanone, and the International =
Consultative=20
Group on Food<BR>Irradiation (ICGFI) for partial financial=20
support.<BR><BR>Bibliography<BR>[only German references given=20
here]<BR><BR>Classen, H. G., P. S. Elias and W. P. Hammes (1987). =20
Toxicological-hygienic<BR>evaluation of food ingredients and additives as =
well=20
as serious<BR>contaminants.<BR>Paul Parey, Berlin and Hamburg.<BR><BR>DGE=
=20
(1996). Nutritional Status of the Federal Republic of=20
Germany.<BR>Nutrition<BR>Report 1996, DGE, Frankfurt am Main, p.=20
43.<BR><BR><BR><BR>"Faillace, Ernie" wrote:<BR><BR>> Dr.=20
Wolke,<BR>><BR>> Could you please indicate a web link to the =
English=20
translation of the<BR>1998<BR>> German study referenced in your March =
7=20
Washington Post article (Nuclear<BR>> Reactions)? Otherwise, =
could you=20
please forward or attach the article in<BR>> reply to this=20
message?<BR>><BR>> Thank you.<BR>><BR>> Ernesto Faillace, =
Eng.D,=20
CHP<BR>> Nuclear Engineer/Health Physicist<BR>> Tetra Tech NUS<BR>>=
; 900=20
Trail Ridge Rd<BR>> Aiken, SC 29803<BR>> (803) 649-7963 x303<BR>> =
(803)=20
642-8454 (fax)<BR>> faillacee@ttnus.com<BR><BR>--<BR>Robert L. =
Wolke<BR><A=20
href=3D"http://www.professorscience.com";>http://www.professorscience.com</A=
><BR><BR><BR></DIV></BODY></HTML>
- --=_134894D5.6A0B6B42--
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