ASTM E1397-91(1998)

NOTICE: This standard has either been superceded and replaced by a new version or discontinued.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.
Designation: E 1397 – 91 (Reapproved 1998)
Standard Practice for
the in vitro Rat Hepatocyte DNA Repair Assay
This standard is issued under the fixed designation E 1397; 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 markedly in many important ways from the patterns of
activation and detoxification that actually occur in hepatocytes.
1.1 This practice covers a typical procedure and guidelines
An extensive literature is available on the use of in vitro
for conducting the rat in vitro hepatocyte DNA repair assay.
hepatocyte DNA repair assays (2, 3, 6, 13-28).
The procedures presented here are based on similar protocols
that have been shown to be reliable (1-6) .
3. Procedure
1.2 Mention of trade names or commercial products are
3.1 Liver Perfusion:
meant only as examples and not as endorsements. Other
3.1.1 All personnel must be knowledgeable in the proce-
suppliers or manufacturers of equivalent products are accept-
dures for safe handling and proper disposal of carcinogens,
able.
potential carcinogens, and radiochemicals. Disposable gloves
1.3 This standard does not purport to address all of the
and lab coats must be worn.
safety concerns associated with its use. It is the responsibility
3.1.2 Any proven technique which allows the successful
of the user of this standard to establish appropriate safety and
isolation and culture of rat hepatocytes can be used. An
health practices and determine the applicability of regulatory
example of one such procedure is given in 3.1.3-3.1.20.
limitations prior to use.
3.1.3 Any strain or sex of rat may be used. The largest
2. Significance and Use database is for male Fischer-344 rats. Young adult animals are
preferred. It is possible that factors such as sex, age, and strain
2.1 Measurement of chemically induced DNA repair is a
of the rat could affect the outcome of the DNA repair
means of assessing the ability of a chemical to reach and alter
experiments. Therefore, for any one series of experiments these
the DNA. DNA repair is an enzymatic process that involves the
variables (including controls) should be kept constant.
recognition and excision of DNA-chemical adduct followed by
3.1.4 Anesthetize the rat by intraperitoneal injection with a
DNA strand polymerization and ligation to restore the original
50-g/mL solution of sodium phenabarbitol (0.2 mL per 100 g
primary structure of the DNA (7). This process can be
body weight) 10 min prior to the perfusion procedure. Other
quantitated by measuring the amount of labeled thymidine
proven anesthetics are also acceptable. Make sure that the
incorporated into the nuclear DNA of cells that are not in
animal is completely anesthetized, so that it feels no pain.
S-phase and is often called unscheduled DNA synthesis (UDS)
3.1.5 Wet the abdomen thoroughly with 70 % ethanol and
(8). Numerous assays have been developed for the measure-
wipe with gauze for cleanliness to discourage loose fur from
ment of chemically induced DNA repair in various cell lines
getting on the liver when the animal is opened.
and primary cell cultures from both rodent and human origin
3.1.6 Make a V-shaped incision through both skin and
(9). The primary rat hepatocyte DNA repair assay developed by
muscle from the center of the lower abdomen to the lateral
Williams (10) has proven to be particularly valuable in
aspects of the rib cage. Do not puncture the diaphragm or cut
assessing the genotoxic activity and potential carcinogenicity
the liver. Fold the skin and attached muscle back over the chest
of chemicals (11), (12). Genotoxic activity is often produced by
to reveal the abdominal cavity.
reactive metabolites of a chemical. The in vitro rat hepatocyte
3.1.7 Place a tube approximately 1 cm in diameter under the
assay provides a system in which a metabolically competent
back to make the portal vein more accessible.
cell is itself the target cell for measured genotoxicity. Most
3.1.8 Move the intestines gently out to the right to reveal the
other short-term tests for genotoxicity employ a rat liver
portal vein. Adjust the tube under the animal so that the portal
homogenate (S-9) for metabolic activation, which differs
vein is horizontal.
3.1.9 Put a suture in place (but not tightened) in the center
of the portal vein and another around the vena cava just above
This practice is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
the right renal branch.
F04.16 on Biocompatibility Test Methods.
3.1.10 Perform perfusions with a peristaltic pump, the
Current edition approved Jan. 25, 1991. Published March 1991.
2 tubing of which is sterilized by circulation of 70 % ethanol
The boldface numbers in parentheses refer to the list of references at the end of
this practice. followed by sterile water. Place a valve in the line so that the
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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 1397 – 91 (1998)
operator may switch from the EGTA solution (see Annex A1) 50-mL centrifuge tube (see Annex A1) using a wide-bore
without disrupting the flow. Keep solutions at a temperature sterile pipet. Some laboratories report successful hepatocyte
that results in a 37°C temperature at the hepatic portal vein. preparations when 3.2.1-3.2.8 are conducted with media at
room temperature or heated to 37°C.
3.1.11 A peristaltic pump with a changeable pump head and
silicone tubing is suitable for performing the perfusion.
3.2.6 Allow the cells to settle on ice for 5 to 10 min until a
distinct interface is seen. Carefully remove and discard the
3.1.12 Begin the flow of the 37°C EGTA solution (see
Annex A1) at 8 mL/min, and run to waste. supernatant by suction.
3.1.13 Cannulate the portal vein with a 20 GA 1 ⁄4-in. 3.2.7 Bring the cells to 50 mL with cold WEI (see Annex
A1). Resuspend the cells by pipeting with a wide-bore pipet.
catheter about 3 mm below the suture. Remove the inner
needle and insert the plastic catheter further to about ⁄3 the Gently pipet the suspension through a 4-ply layer of sterile
gauze into a sterile 50-mL centrifuge tube.
length of the vein and tie in place by the suture. Blood should
emerge from the catheter. Insert the tube with the flowing
3.2.8 Centrifuge the cells at 50 times gravity for 5 min and
EGTA (see Annex A1) in the catheter (avoid bubbles) and tape
discard the supernatant. Gently resuspend the pellet in ice-cold
in place.
WEI (see Annex A1) with a wide-bore pipet.
3.1.14 Immediately cut the vena cava below the right renal
3.2.9 Some laboratories prefer to keep the cells on ice until
branch and allow the liver to drain of blood for 1.5 min. The
ready for use, while others keep them at room temperature.
liver should rapidly clear of blood and turn a tan color. If all
Cells should be used as soon as possible, preferably within 1 h.
lobes do not clear uniformly, the catheter has probably been
3.2.10 Determine viability and cell concentration by the
inserted too far into the portal vein.
method of trypan blue exclusion. The preparation should be
3.1.15 Tighten the suture around the vena cava and increase
primarily a single-cell suspension with a viability of over 60 %
the flow to 20 mL/min for 2 min. The liver should swell at this
for control cultures. With practice and the proper technique,
point. In some cases gentle massaging of the liver or adjusting
viabilities of about 90 % can routinely be obtained. Attachment
the orientation of the catheter may be necessary for complete
of the cells to the substrate is an active process. Thus, if a
clearing. At this point the vena cava above the suture may be
sufficient number of cells attach to conduct the experiment, it
clipped to release some of the pressure in the liver.
is a further indication of the viability of the culture.
3.1.16 Switch the flow to the 37°C collagenase solution for
3.2.11 Place a 25-mm round plastic coverslip into each well
12 min. During this period, cover the liver with sterile gauze
of 6-well culture dishes. 10.5 by 22-mm plastic coverslips and
wetted with sterile saline or WEI (see Annex A1) and place a
26 by 33-mm eight-chamber culture dishes can also be used.
40-W lamp 2 in. above the liver for warming. It is valuable to
Be sure to keep the proper side up as marked on the package.
screen each new batch of collagenase to be ensured that it will
Add 4 mL of WEC (see Annex A1) to each well. Hepatocytes
function properly.
will not attach to glass unless the slides have been boiled. The
3.1.17 Allow the perfusate to flow onto the paper and collect
use of collagen-coated thermanox coverslips improves cell
by suction into a vessel connected by means of a trap to the
attachment and morphology.
vacuum line.
3.2.12 These procedures yield preparations consisting pri-
3.1.18 After the perfusion is over, remove the catheter and
marily of hepatocytes. Approximately 400 000 viable cells are
gauze. Carefully remove the liver by cutting away the mem-
seeded into each well and distributed over the coverslip by
branes connecting it to the stomach and lower esophagus,
shaking or stirring gently with a plastic 1-mL pipet. Glass
cutting away the diaphragm, and cutting any remaining attach-
pipettes can scratch the coverslips.
ments to veins or tissues in the abdomen.
3.2.13 Incubate the cultures for 90 to 120 min in a 37°C
3.1.19 Hold the liver by the small piece of attached dia-
incubator with 5 % CO and 95 % relative humidity, to allow
phragm and rinse with sterile saline or WEI (see Annex A1).
the cells to attach.
3.1.20 Place the liver in a sterile petri dish and take to a
3.3 Labeling the Cultures:
sterile hood to prepare the cells.
3.3.1 After the attachment period, wash the cultures once
3.2 Preparation of Hepatocyte Cultures:
with 4 mL WEI (see Annex A1) per well to remove unattached
3.2.1 Place the perfused liver in a 60-mm petri dish and
cells and debris. This is done by tilting the culture slightly,
rinse with 37°C WEI (see Annex A1). Remove extraneous
aspirating the media, and adding the fresh media at 37°C. Be
tissues (fat, muscle, and so forth).
careful not to direct the stream from the pipet directly onto the
3.2.2 Place the liver in a clean petri dish and add 30 mL of
cells.
fresh collagenase solution (see Annex A1) at 37°C.
3.3.2 Prepare chemical solutions in H-thymidine solution
3.2.3 Carefully make several incisions in the capsule of each
(WEI containing 10 μCi/mL H-thymidine) (see Annex A1).
lobe of the liver. Large rips in the capsule lead to large
Serial dilutions are generally employed. If employed, solvents
unusable clumps of hepatocytes.
for the test substance, such as dimethyl sulfoxide (DMSO) or
3.2.4 Gently comb out the cells, constantly swirling the liver ethanol, should not exceed a 1 % final concentration. Most
while combing. A sterile metal dog-grooming comb with teeth
investigators try to limit the DMSO concentration to at or
spaced from 1 to 3 mm apart, or a hog bristle brush works well. below 0.5 %, because of borderline toxic effects on some
3.2.5 When only fibrous and connective tissue remain, hepatocyte cultures at DMSO concentrations of 1 %. Both
remove and discard the remaining liver. Add 20 mL cold WEI medium alone and solvent controls should be included in the
(see Annex A1) and transfer the cell suspension to a sterile experimental design. Concentrations of the test substance
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 1397 – 91 (1998)
should be chosen that go just beyond cytotoxicity to about 3.4.4 Mount a 50-mL disposable plastic beaker with tape
1000-fold below the cytotoxic concentration. Cytotoxicity can into a slightly larger jar full of water. Place this assembly into
be determined by trypan blue dye exclusion or lactic dehydro- a 42°C water bath and allow to reach the bath temperature.
genase (LDH) release of the cultures or by morphological 3.4.5 Kodak NTB-2 emulsion is most commonly used. The
examination of the fixed and stained cells at the end of the emulsion may be used undiluted or can be diluted toa1to1
experiment. Typical concentration ranges are from 10 to 0.001 ratio with distilled water. If the emulsion is diluted, take care to
mM. Relatively insoluble substances should be tested up to use double distilled or ultrapure water; thoroughly mix the
their limit of solubility. Freely soluble, nontoxic chemicals solution but avoid formation of air bubbles. Undiluted emul-
should not be tested at concentrations beyond 10 mM. A dose sion saves a step and provides slightly higher grain counts.
of 10 mM dimethylnitrosamine (DMN) is required to produce Melt emulsion in a 37°C incubator for at least 3 h. Gently pour
a strong DNA repair response in the assay. In contrast, 40 to 50 mL of the emulsion into the 50-mL disposable beaker.
1,6-dinitropyrene induces DNA repair at concentrations as low The unused portion can be resealed and stored under refrig-
as 0.00005 mM. eration. If one of the Ilford “K” series of photographic
emulsions is used, it must not be liquefied and regelled.
3.3.3 Remove the WEI (see Annex A1) and replace with 2
mL of H-thymidine solution (see Annex A1) containing the 3.4.6 Dip a test slide. Briefly turn on the red safelight and
hold the slide up to it to make sure that there is enough
dissolved test chemical. Place the cultures in the incubator for
16 to 24 h. During this period the compound may be metabo- emulsion in the cup to cover the cells, and that there are no
bubbles in the emulsion. Air bubbles can be removed from the
lized. If DNA damage occurs, it will be repaired, resulting in
incorporation of the H-thymidine (see Annex A1). surface of the emulsion by skimming the surface with a glass
slide. Turn off the safelight.
3.3.4 Wash cultures twice with 4 mL WEI (see Annex A1)
3.4.7 Dip each group of slides by lowering them into the cup
per well.
until they touch the bottom. Pull the slides out of the emulsion
3.3.5 The remainder of these procedures are done with
with a smooth action to a 5-s count. Touch the bottom ends of
solutions at room temperature. Replace the medium with 4 mL
the slides to a pad of paper towels to remove the bead of
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