ASTM E1398-91(1998)
(Practice)Standard Practice for the in vivo Rat Hepatocyte DNA Repair Assay
Standard Practice for the in vivo Rat Hepatocyte DNA Repair Assay
SCOPE
1.1 This practice covers a typical procedure and guidelines for conducting the rat in vivo hepatocyte DNA repair assay. The procedures presented here are based on similar protocols that have been shown to be reliable (1, 2, 3, 4, 5).
1.2 Mention of trade names or commercial products are meant only as examples and not as endorsements. Other suppliers or manufacturers of equivalent products are acceptable.
1.3 This standard does not purport to address all of the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Standards Content (Sample)
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
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Designation: E 1398 – 91 (Reapproved 1998)
Standard Practice for
the in vivo Rat Hepatocyte DNA Repair Assay
This standard is issued under the fixed designation E 1398; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope occur in hepatocytes. An extensive literature is available on the
use of in vitro DNA repair assays (9-19).
1.1 This practice covers a typical procedure and guidelines
2.2 A further advance was the development of an in vivo rat
for conducting the rat in vivo hepatocyte DNA repair assay. The
hepatocyte DNA repair assay in which the test chemical is
procedures presented here are based on similar protocols that
administered to the animal and the resulting DNA repair is
have been shown to be reliable (1, 2, 3, 4, 5).
assessed in hepatocytes isolated from the treated animal (20).
1.2 Mention of trade names or commercial products are
Numerous systems now exist to measure chemically induced
meant only as examples and not as endorsements. Other
DNA repair in specific tissues in the whole animal (4). The
suppliers or manufacturers of equivalent products are accept-
average of in vivo assays is that they reflect the complex
able.
patterns of uptake, distribution, metabolism, detoxification,
1.3 This standard does not purport to address all of the
and excretion that occur in the whole animal. Further, factors
safety concerns associated with its use. It is the responsibility
such as chronic exposure, sex differences, and different routes
of the user of this standard to establish appropriate safety and
of exposure can be studied with these systems. This is
health practices and determine the applicability of regulatory
illustrated by the potent hepatocarcinogen 2,6-dinitrotoluene
limitations prior to use.
(DNT). Metabolic activation of 2,6-DNT involves uptake,
2. Significance and Use
metabolism by the liver, excretion into the bile, reduction of
the nitro group by gut flora, readsorption, and further metabo-
2.1 Measurement of chemically induced DNA repair is a
lism by the liver once again to finally produce the ultimate
means of assessing the ability of a chemical to reach and alter
genotoxicant (21). Thus, 2,6-DNT is negative in the in vitro
the DNA. DNA repair is an enzymatic process that involves
hepatocyte DNA repair assay (22) but is a very potent inducer
recognition and excision of DNA-chemical adducts, followed
of DNA repair in the in vivo DNA repair assay (23, 24).A
by DNA strand polymerization and ligation to restore the
problem with tissue-specific assays is that they may fail to
original primary structure of the DNA (6). This process can be
detect activity of compounds that produce tumors in other
quantitated by measuring the amount of labeled thymidine
target tissues. For example, no activity is seen in the in vivo
incorporated into the nuclear DNA of cells that are not in
DNA repair assay with the potent mutagen benzo(a)pyrene
S-phase and is often called unscheduled DNA synthesis (UDS)
(BP), probably because limited tissue distribution and greater
(7). Numerous assays have been developed for the measure-
detoxification in the liver yields too few DNA adducts to
ment of chemically induced DNA repair in various cell lines
produce a measurable response (3). In contrast, BP is readily
and primary cell cultures from both rodent and human origin
detected in the less tissue-specific in vitro hepatocyte DNA
(4). The primary culture rat hepatocyte DNA repair assay has
repair assay (11). An extensive literature exists on the use of
proven to be particularly valuable in assessing the genotoxic
the in vivo hepatocyte DNA repair assay (1-3, 5, 9, 25-33).
activity of chemicals (8). Genotoxic activity often results from
metabolites of a chemical. The in vitro rat hepatocyte assay
3. Procedure
provides a system in which a metabolically competent cell is
3.1 Treatment:
also the target cell. Most other in vitro short-term tests for
3.1.1 All personnel must be knowledgeable in the proce-
genotoxicity employ a rat liver homogenate (S-9) for metabolic
dures for safe handling and proper disposal of carcinogens,
activation, which differs markedly in many important ways
potential carcinogens, and radiochemicals. Disposable gloves
from the patterns of activation and detoxification that actually
and lab coats must be worn.
3.1.2 Any strain or sex of rat may be used. The largest
This practice is under the jurisdiction of ASTM Committee F04 on Medical and
database is for male Fischer-344 rats. Young adult animals are
Surgical Materials and Devices and is the direct responsibility of Subcommittee
preferred. It is possible that factors such as sex, age, and strain
F04.16 on Biocompatibility Test Methods.
of the rat could affect the outcome of the DNA repair
Current edition approved Jan. 25, 1991. Published March 1991.
The boldface numbers in parentheses refer to the list of references found at the
end of this practice.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1398 – 91 (1998)
experiments. Therefore, for any one series of experiments 3.2.2 DMN exhibits a maximum UDS response 1 h after
(including controls) these variables should be kept constant. treatment. However, the response remains elevated and mea-
surable for at least 16 h after treatment. More commonly,
3.1.3 Administration is usually by gavage with chemicals
however, chemicals (for example, 2,6-DNT and
dissolved or suspended in an appropriate vehicle such as water
2-acetylaminofluorene (2-AAF)) show a peak response 12 to
or corn oil, depending on solubility. An advantage of the assay
16 h post-treatment.The time from treatment to perfusion may
is that various routes of administration may be chosen. Thus,
be varied to obtain a time course of induced repair. The routine
chemicals may also be administered by intraperitoneal injec-
protocol for primary screening involves a time point between
tion or inhalation or in the diet. For gavage administration, 0.2
12 and 16 h with an optional time point between 1 and 4 h.
to 1.0 mL of test chemical solution is administered per 100 g
3.2.3 Anesthetize the rat by intraperitoneal injection with a
body weight. Controls receive the appropriate vehicle solution.
Stock corn oil should be replaced with fresh monthly. 50-mg/mL solution of sodium pentobarbital (0.2 mL per 100 g
body weight) 10 min prior to the perfusion procedure. Other
3.1.4 For DNA repair studies, animals may be taken off feed
proven anesthetics are also acceptable. Make sure that the
for a few hours prior to sacrifice to make the process of
animal is completely anesthetized, so that it feels no pain.
perfusion a little easier with less food in the stomach. The
3.2.4 Secure the animal with the ventral surface up on
period without food should never exceed 12 h because of the
absorbent paper attached to a cork board. Fold the paper in on
possibility of altered metabolism or uptake. Water should be
each edge to contain perfusate overflow. Thoroughly wet the
continuously available.
abdomen with 70 % ethanol and wipe with gauze for cleanli-
3.1.5 Dose selection will depend on the characteristics of
ness and to discourage loose fur from getting on the liver when
each chemical and the purpose of the experiment. If one is
the animal is opened.
investigating whether a chemical can produce a genotoxic
3.2.5 Make a V-shaped incision through both skin and
effect in the animal, even at massive doses and by routes of
muscle from the center of the lower abdomen to the lateral
administration that may overwhelm natural defense mecha-
aspects of the rib cage. Do not puncture the diaphragm or cut
nisms, then high doses (such as the LD50, or higher) that do
the liver. Fold back the skin and attached muscle over the chest
not kill the animal before the 16-h sacrifice point may be
to reveal the abdominal cavity.
employed. Even in such a case, doses above 1000-mg/kg body
3.2.6 Place a tube approximately 1 cm in diameter under the
weight are not recommended. In some instances hepatotoxicity
back to make the portal vein more accessible.
at high doses may result in inhibition of cell attachment or
DNA repair. More commonly, the purpose of employing the 3.2.7 Move the intestines gently out to the right to reveal the
whole animal is to evaluate the genotoxic effects of realistic portal vein. Adjust the tube under the animal so that the portal
exposures and routes of administration in the target tissue. In vein is horizontal.
this case, doses above 500 mg/kg and the intraperitoneal route
3.2.8 Put a suture in place (but not tightened) in the center
of administration are not recommended. The usual range of of the portal vein and another around the vena cava just above
doses is from 10 to 500 mg compound per kilogram body
the right renal branch.
weight. Target doses with a new compound are usually the
3.2.9 Perform perfusions with a peristaltic pump, the tubing
LD50 and 0.2 3 the LD50, with 500 mg/kg as an upper limit.
of which is sterilized by circulation of 70 % ethanol followed
The potent mutagen dimethylnitrosamine (DMN) (often used
by sterile water. Place a valve in the line so that the operator
as a positive control) can be detected with doses as low as 1
may switch from the EGTA solution to the collagenase solution
mg/kg. Normally, an initial range finding experiment is con-
without disrupting the flow. Solutions should be kept at a
ducted using single animals to cover a range of times and
temperature that results in a 37°C temperature at the hepatic
doses. If a positive response is seen, additional experiments are
portal vein.
conducted to establish the dose-response relationship. If no
3.2.9.1 A peristaltic pump with a chargeable pump head and
response is seen, the highest dose(s) is repeated. The final
silicone tubing is suitable for performing the perfusion.
report should contain results from at least three animals per
3.2.9.2 Begin the flow of the 37°C EGTA solution at 8
datapoint.
mL/min and run to waste.
3.1.6 Thus far, no examples have been seen of a compound 1
3.2.10 Cannulate the portal vein with a 20 GA 1 ⁄4-in.
that produces a UDS response in female rats but not males. For
catheter about 3 mm below the suture. Remove the inner
those cases where a comparison has been made, males respond 1
needle and insert the plastic catheter further to about ⁄3 the
more strongly than females in this assay. Thus, for the purpose
length of vein and tie in place by the suture. Blood should
of routine screening only male rats need to be used.
emerge from the catheter. Insert the tube with the flowing
3.1.7 Treated animals should be maintained in a ventilated
EGTA in the catheter (avoid bubbles) and tape in place.
area or other suitable location to prevent possible human
3.2.11 Immediately cut the vena cava below the right renal
exposure to expired chemicals. Contaminated cages, bedding,
branch and allow the liver to drain of blood for 1.5 min. The
excreta, and carcasses should be disposed of safely according
liver should rapidly clear of blood and turn a tan color. If all
to standard published procedures.
lobes do not clear uniformly, the catheter has probably been
3.2 Liver Perfusion: inserted too far into the portal vein.
3.2.1 Any proven technique which allows the successful 3.2.12 Tighten the suture around the vena cava and increase
isolation and culture of rat hepatocytes can be used. An the flow to 20 mL/min for 2 min. The liver should swell at this
example of one such procedure is given in 3.2.2-3.2.17. point. In some cases gentle massaging of the liver or adjusting
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1398 – 91 (1998)
the orientation of the catheter may be necessary for complete for control cultures. With practice and the proper technique,
clearing. At this point the vena cava above the suture may be viabilities of about 90 % can routinely be obtained. Attachment
clipped to release some of the pressure in the liver. of the cells to the substrate is an active process. Thus, if a
sufficient number of cells attach to conduct the experiment, this
3.2.13 Switch the flow to the 37°C collagenase solution for
12 min. During this period, cover the liver with sterile gauze is a further indication of the viability of the culture.
wetted with sterile saline or WEI (see Annex A1) and place a 3.3.10 Place a 25-mm round plastic coverslip into each well
40-W lamp 2 in. above the liver for warming. It is valuable to
of 6-well culture dishes. 10.5 by 22-mm plastic coverslips and
screen each new batch of collagenase to be ensured that it will 26 by 33-mm eight-chamber culture dishes can also be used.
function properly. Be sure to keep the proper side up as marked on the package.
3.2.14 Allow the perfusate to flow onto the paper and collect Four millilitres of WEC (see Annex A1) are added to each well.
Hepatocytes will not attach to glass unless the slides have been
by suction into a vessel connected by means of a trap to the
vacuum line. The perfusate should be disposed of as hazardous boiled. The use of collagen-coated thermanox coverslips im-
proves cell attachment and morphology.
waste.
3.2.15 After the perfusion is completed, remove the catheter 3.3.10.1 These procedures yield preparations consisting pri-
and gauze. Remove the liver carefully by cutting away the marily of hepatocytes. Approximately 400 000 viable cells are
seeded into each well and distributed over the coverslip by
membranes connecting it to the stomach and lower esophagus,
cutting away the diaphragm, and cutting any remaining attach- shaking or stirring gently with a plastic 1-mL pipet. Glass
pipettes can scratch the coverslips.
ments to veins or tissues in the abdomen.
3.2.16 Hold the liver by the small piece of attached dia- 3.3.11 Incubate the cultures for 90 to 120 min in a 37°C
phragm and rinse with sterile saline or WEI (see Annex A1). incubator with 5 % CO and 95 % relative humidity to allow
3.2.17 Place the liver in a sterile petri dish and take to a the cells to attach.
sterile hood to prepare the cells. 3.4 Labeling the Cultures:
3.3 Preparation of Hepatocyte Cultures: 3.4.1 After the 90-min attachment, wash cultures once with
4 mL W
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