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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well-documented

Data source

Reference
Reference Type:
publication
Title:
Cross-Linking of DNA in Liver and Testes of Rats Fed 1,3-Propanediol
Author:
Summerfield F.W., Tappel A.L.
Year:
1984
Bibliographic source:
Chemical-Biological Interactions, 1984, 50, 87-96

Materials and methods

Principles of method if other than guideline:
Study of cross-Linking of DNA in Liver and Testes of Rats Fed 1,3-Propanediol.
1,3-Propanediol (PAD) was fed to rats for 15 weeks, and its effects on hepatic and testicular DNA were studied. The control rats were fed a casein-based diet that contained 10% tocopherol-stripped corn oil with 30 IU of d, l-alpha-tocopherol acetate/kg; the experimental rats were fed the same diet
with 500 ppm of PAD.
GLP compliance:
no
Type of assay:
other: Cross-Linking of DNA in Liver and Testes of Rats

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
1,3-propanediol obtained from Eastman Chemical Company was distilled twice before use; only the fraction that distilled at over 200°C was used.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Inc.
- Age at study initiation: 3 weeks.

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with Casein-based diet (Teklad Test Diets).
Duration of treatment / exposure:
5, 10 and 15 weeks.
Frequency of treatment:
Daily in diet.
Post exposure period:
None.
Doses / concentrations
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
3 x 3 males (3 each killed after 5, 10 and 15 weeks).
Control animals:
yes, plain diet
Positive control(s):
None.

Examinations

Tissues and cell types examined:
DNA extracted from liver and testes.
Details of tissue and slide preparation:
Animals and diets:
Male Sprague-Dawley rats, obtained at 3 weeks of age from Simonsen
Laboratories, Inc., were fed a casein-based diet (Teklad Test Diets)
that contained 10% tocopherol-stripped corn oil, mineral mix 4179 and all
essential nutrients, including 30 IU of d,l-a-tocopherol acetate/kg. Control
rats were fed this diet without 1,3-propanediol (PAD); the experimental rats were fed this
diet with 500 ppm PAD.
Three rats in each group were decapitated at 5, 10 and 15 weeks. Their
livers and testes were immediately removed and placed on ice. The epididymides
were trimmed from the testes to avoid contamination by spermatozoa.
One testicle from each rat was used for measurement of lipid-soluble fluorophores.
This tissue was minced and extracted with 10 ml of chloroform/
methanol (2: l ) / g of tissue by grinding in a motor-driven glass-Teflon homogenizer
for 90 s at 37°C. The homogenate was centrifuged, and the supernatant
was filtered through glass wool and washed with an equal volume of
water. After separation of the phases, the fluorescence of the organic phase
was measured at 365 nm excitation and 460 nm emission. Fluorescence
values were normalized to a fluorescence of 100 at 350 nm excitation and
450 nm emission for 10~s M quinine sulfate in 0.1 N H2S04 .

Isolation of DNA:
The DNA was isolated from the liver and one testicle of each rat.
The tissues were minced and homogenized in 4 volumes of 10 mM sodium
phosphate buffer (pH 6.8) that contained 0.35 M sucrose and 1 mM CaCl2
in a motor-driven glass-Teflon homogenizer at high speed for 25 strokes.
Immediately before homogenization, 0.01 vol. of a solution of 0.1 M free
d, l-a-tocopherol in ethanol was added. The homogenate was layered over
the same buffer that contained 1.75 M sucrose for liver and 1.55 M sucrose
for testes, and the nuclei were isolated by centrifugation at 70 000 X g for
2 h. The nuclear pellet was washed twice by resuspending it in 10 mM
sodium phosphate buffer (pH 7.5) that contained 75 mM NaCl and 5 mM
EDTA, and centrifuging at 1000 X g for 5 min. The nuclei were digested
with 0.6 ml of the protease K solution described above, in a total volume
of 3 ml for 1216 h. The digest was extracted twice with an equal volume
of 85% phenol that contained 15 mM Tris (pH 8). The aqueous layer was
applied to a 1 X 25-cm column of Sepharose 6B and eluted with 10 mM
sodium phosphate buffer (pH 7). Absorbance was monitored at 254 nm. The
fractions that contained the DNA were pooled, concentrated by lyophilization
to 0.51 ml and stored at 20°C.
To prevent contamination of the nuclei by cytoplasm and various organelles
and to minimize damage to the nuclei and DNA by osmotic effects,
the optimum concentration of sucrose in the gradient buffer for both liver
and testes was first determined. Protein was determined by the Bradford
method and DNA by reaction with diphenylamine. Cytoplasmic
contamination was determined by measuring lactic dehydrogenase activity.
Lysosomal contamination was determined by measuring acid phosphatase
activity at pH 5 with p-hitrophenyl phosphate as substrate. Mitochondrial
contamination was determined by measurement of succinic
dehydrogenase activity.

Production of malondialdehyde (MDA) from PAD by tissue homogenates:
After the rats had been fed PAD for 15 weeks, homogenates of the livers
and testes of two rats of each group were prepared by the procedure described
above, except that a-tocopherol was added in oleic acid. Each
reaction mixture contained 100 mg of homogenized tissue, 20 mM JV-ethylmorpholine
buffer (pH 9), 10 mM sodium pyruvate, 20 nM lactic dehydrogenase,
0.1 mM NAD and either 0 or 10 mM PAD. Each reaction was done
in duplicate at 37°C for 3 h. Then, to each mixture was added 3 ml of
10 mM 2-thiobarbituric acid in 25 mM sodium phosphate buffer (pH 7)
and 1 ml of 50% trichloroacetic acid. The mixture was heated for 1 h at
95°C, clarified by centrifugation, and the absorbance of the supernatant
was measured at 532 nm. When this method was used, 10 nmol of MDA
produced an absorbance of 0.3.

Tryptophan bound to DNA:
Tryptophan is an amino acid with high native fluorescence, thus it provides
a very sensitive marker for the binding of proteins to DNA. Bound
tryptophan was measured by fluorescence at 280 nm excitation and 350 nm
emission. The specific fluorescence intensity of tryptophan was determined
using histone standards. Because DNA quenches the fluorescence of tryptophan
by absorbing the exciting light, the effect of the concentration of
DNA on this fluorescence was determined; bovine serum albumin was used
because DNA precipitated the histone. The fluorescence in the samples of
DNA, or diluted aliquots thereof, isolated from the rat tissues was measured
spectrophotofluorometrically, and the fluorescence was corrected for
dilution and quenching. The values calculated from these measurements
are expressed as bound tryptophan residues per 1 0 s base pairs of DNA.

Template activity of DNA:
The 300-jul reaction mixture for measuring DNA template activity contained
10 jug of DNA, 40 mM Tris (pH 8), 4 mM MgCl2 , 100 mM KCl,
0.4 mM dithioerythritol, 0.20.5 /xl of the stock solution of RNA polymerase
and 0.2 mM ATP, CTP, GTP and UTP. The specific activity of the
[3H]UTP varied from 1 - 3 X 104 cpm/nmol. Calf thymus DNA was used
as a control to normalize for differences in the specific activity of [ 3H]UTP
or polymerase used in assays done at different times. After 34 h at 37°C,
the reaction was terminated by the addition of 0.5 ml of 50 mM sodium
pyrophosphate, pH 8, that contained 0.2 M sodium dodecyl sulfate and
2 mg/ml of bovine serum albumin and of RNA. The mixture was cooled
in an ice bath and 0.2 ml of 50% trichloroacetic acid was added. After
23 h, the precipitate was collected on nitrocellulose filters, washed extensively
with ice-cold 10% trichloroacetic acid and air dried. The radioactivity
on the filters was counted by liquid scintillation.
Statistics:
The significance of differences between any two groups was determined
by the Student's two-tailed t-test. P-values <0.05 were considered significant.

Results and discussion

Additional information on results:
Purity of nuclei:
The effect of the concentration of sucrose in the gradient buffer on the
purity of nuclei isolated from rat liver by centrifugation was determined.
Concentrations of sucrose greater than 1.5 M prevented nearly all cytoplasmic
and lysosomal contamination. When 1.6 M sucrose was used, there
was still noticeable mitochondrial contamination as measured by succinic
dehydrogenase activity. This contamination was eliminated by using 1.7 M
sucrose in the gradient buffer. The DNA content was not affected by higher
concentrations of sucrose and, since no further improvement in the preparation
was observed, 1.75 M was chosen as the optimal concentration of
sucrose for isolating nuclei from liver. The sucrose concentration used to
isolate nuclei from testes was 1.55 M, primarily because a higher sucrose
concentration caused a significant loss of nuclei.

Production of MDA by homogenates:
Liver homogenates from both control and PAD-fed rats converted PAD
to MDA at the rate of about 5.6 nmol/h/100 mg of tissue. Testicular homogenates
from either group of rats had little or no ability to catalyze this
conversion.

Lipid-soluble testicular fluorophores:
At 15 weeks fluorescence of testicular extracts of
the experimental rats was higher than that for the control rats (P = 0.03).
The fluorophores did not increase in the control rat testes between 5 and
15 weeks; although the fluorophores in the experimental rat testes were
approximately twice as high at 10 and 15 weeks as at 5 weeks, the differences
were not significant.

DNA-bound tryptophan:
The amount of tryptophan bound to the hepatic DNA of PAD-fed rats at 15 weeks
was higher than that of the control group (P = 0.038) and higher than
that from rats fed PAD for 5 weeks (P = 0.05). The amount of tryptophan
bound to the testicular DNA from the two groups of rats did not
differ at any time point.

Template activity of DNA:
The template activity of the hepatic and testicular DNA of each group
of rats did not exceed 6070% of that of the calf thymus DNA that was
used as a control. As estimated by a comparison of incorporated
radioactivity, the hepatic DNA of PAD-fed rats had lower template activity
than that of control rats at 10 (P = 0.055) and 15 weeks (P = 0.048). By
comparison with the template activity of the calf thymus DNA control,
the hepatic DNA template activity was lower at 15 weeks (47%) than it was
at 10 weeks (58%). The differences between the template activities of the
testicular DNAs of the two groups were neither different at any time point
nor different between any two time points.

Cross-links in DNA:
The amounts of both DNA-protein and interstrand DNA cross-links in the
hepatic DNA of PAD-fed rats were higher than those of the control rats at
10 and 15 weeks. There were also small increases in the amounts
of DNA-protein and interstrand cross-links in the testicular DNA of the
PAD-fed rats at 15 weeks. Among time points, there were no differences in
the cross-linking of hepatic or testicular DNA of the control rats. For the
hepatic DNA of PAD-fed rats, the DNA-protein cross-links and the interstrand
DNA cross-links both increased from 5 to 10 weeks and from 10 to
15 weeks.

Applicant's summary and conclusion

Conclusions:
The results are consistent with the conclusion that 1,3-Propanediol was converted to malondialdehyde in vivo and that malondialdehyde is the reactive species that caused the observed biological damage
Executive summary:

1,3-Propanediol (PAD) was fed to rats for 15 weeks, and its effects on hepatic and testicular DNA were studied. The control rats were fed a casein-based diet that contained 10% tocopherol-stripped corn oil with 30 IU of d,l-alpha-tocopherol acetate/kg; the experimental rats were fed the same diet with 500 ppm of PAD. Homogenates prepared from the livers of each group of rats converted 1,3-propanediol to malondialdehyde (MDA) with equal efficacy, but homogenates of testes did not catalyze this conversion. After 10-15 weeks of feeding the diets, the hepatic DNA of the rats fed PAD had less template activity, more bound tryptophan and more DNA-protein and interstrand DNA cross-links than that of the control rats. As measured by template activity and bound tryptophan, testicular DNA of the experimental rats was not different from that of the control rats; however, there was slightly more cross-linking in the testicular DNA of experimental rats than in that of control rats. Testes of the experimental rats contained more lipid-soluble fluorophores than did those of the control rats. The results are consistent with the conclusion that PAD was converted to MDA in vivo and that MDA is the reactive species that caused the observed biological damage.