ASTM E1397-91(2003)

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Designation:E1397–91(Reapproved 2003)
Standard Practice for
In Vitro Rat Hepatocyte DNA Repair Assay
This standard is issued under the fixed designation E1397; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope homogenate (S-9) for metabolic activation, which differs
markedly in many important ways from the patterns of
1.1 This practice covers a typical procedure and guidelines
activationanddetoxificationthatactuallyoccurinhepatocytes.
for conducting the rat in vitro hepatocyte DNA repair assay.
An extensive literature is available on the use of in vitro
The procedures presented here are based on similar protocols
hepatocyte DNA repair assays (2, 3, 6, 13-28).
that have been shown to be reliable (1-6) .
1.2 Mention of trade names or commercial products are
3. Procedure
meant only as examples and not as endorsements. Other
3.1 Liver Perfusion:
suppliers or manufacturers of equivalent products are accept-
3.1.1 All personnel must be knowledgeable in the proce-
able.
dures for safe handling and proper disposal of carcinogens,
1.3 This standard does not purport to address all of the
potential carcinogens, and radiochemicals. Disposable gloves
safety concerns associated with its use. It is the responsibility
and lab coats must be worn.
of the user of this standard to establish appropriate safety and
3.1.2 Any proven technique which allows the successful
health practices and determine the applicability of regulatory
isolation and culture of rat hepatocytes can be used. An
limitations prior to use.
example of one such procedure is given in 3.1.3-3.1.20.
2. Significance and Use 3.1.3 Any strain or sex of rat may be used. The largest
database is for male Fischer-344 rats.Young adult animals are
2.1 Measurement of chemically induced DNA repair is a
preferred. It is possible that factors such as sex, age, and strain
means of assessing the ability of a chemical to reach and alter
of the rat could affect the outcome of the DNA repair
theDNA.DNArepairisanenzymaticprocessthatinvolvesthe
experiments.Therefore,foranyoneseriesofexperimentsthese
recognitionandexcisionofDNA-chemicaladductfollowedby
variables (including controls) should be kept constant.
DNAstrand polymerization and ligation to restore the original
3.1.4 Anesthetize the rat by intraperitoneal injection with a
primary structure of the DNA (7). This process can be
50-g/mL solution of sodium phenabarbitol (0.2 mL per 100 g
quantitated by measuring the amount of labeled thymidine
body weight) 10 min prior to the perfusion procedure. Other
incorporated into the nuclear DNA of cells that are not in
proven anesthetics are also acceptable. Make sure that the
S-phaseandisoftencalledunscheduledDNAsynthesis(UDS)
animal is completely anesthetized, so that it feels no pain.
(8). Numerous assays have been developed for the measure-
3.1.5 Wet the abdomen thoroughly with 70% ethanol and
ment of chemically induced DNA repair in various cell lines
wipe with gauze for cleanliness to discourage loose fur from
and primary cell cultures from both rodent and human origin
getting on the liver when the animal is opened.
(9).TheprimaryrathepatocyteDNArepairassaydevelopedby
3.1.6 Make a V-shaped incision through both skin and
Williams (10) has proven to be particularly valuable in
muscle from the center of the lower abdomen to the lateral
assessing the genotoxic activity and potential carcinogenicity
aspects of the rib cage. Do not puncture the diaphragm or cut
ofchemicals(11),(12).Genotoxicactivityisoftenproducedby
theliver.Foldtheskinandattachedmusclebackoverthechest
reactive metabolites of a chemical. The in vitro rat hepatocyte
to reveal the abdominal cavity.
assay provides a system in which a metabolically competent
3.1.7 Placeatubeapproximately1cmindiameterunderthe
cell is itself the target cell for measured genotoxicity. Most
back to make the portal vein more accessible.
other short-term tests for genotoxicity employ a rat liver
3.1.8 Movetheintestinesgentlyouttotherighttorevealthe
portal vein.Adjust the tube under the animal so that the portal
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
vein is horizontal.
Surgical Materials and Devices and is the direct responsibility of Subcommittee
3.1.9 Put a suture in place (but not tightened) in the center
F04.16 on Biocompatibility Test Methods.
of the portal vein and another around the vena cava just above
Current edition approved Sept. 10, 2003. Published September 2003. Originally
approved in 1991. Last previous edition approved in 1998 as E1397–91 (1998).
the right renal branch.
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this practice.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1397–91 (2003)
3.1.10 Perform perfusions with a peristaltic pump, the 3.2.5 When only fibrous and connective tissue remain,
tubing of which is sterilized by circulation of 70% ethanol remove and discard the remaining liver.Add 20 mLcold WEI
followed by sterile water. Place a valve in the line so that the (see Annex A1) and transfer the cell suspension to a sterile
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 Aperistalticpumpwithachangeablepumpheadand
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
3.1.12 Begin the flow of the 37°C EGTA solution (see distinct interface is seen. Carefully remove and discard the
supernatant by suction.
Annex A1) at 8 mL/min, and run to waste.
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
catheter about 3 mm below the suture. Remove the inner A1). Resuspend the cells by pipeting with a wide-bore pipet.
needle and insert the plastic catheter further to about ⁄3 the Gently pipet the suspension through a 4-ply layer of sterile
length of the vein and tie in place by the suture. Blood should gauze into a sterile 50-mL centrifuge tube.
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(seeAnnexA1) in the catheter (avoid bubbles) and tape
discardthesupernatant.Gentlyresuspendthepelletinice-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
Cellsshouldbeusedassoonaspossible,preferablywithin1h.
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 Tightenthesuturearoundthevenacavaandincrease
primarilyasingle-cellsuspensionwithaviabilityofover60%
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
viabilitiesofabout90%canroutinelybeobtained.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 Placea25-mmroundplasticcoverslipintoeachwell
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 (seeAnnexA1) to each well. Hepatocytes
function properly.
will not attach to glass unless the slides have been boiled. The
3.1.17 Allowtheperfusatetoflowontothepaperandcollect
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 400000 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 mLWEI (seeAnnexA1) 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 Carefullymakeseveralincisionsinthecapsuleofeach
(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 Gentlycomboutthecells,constantlyswirlingtheliver ethanol, should not exceed a 1% final concentration. Most
while combing.Asterile metal dog-grooming comb with teeth investigators try to limit the DMSO concentration to at or
spacedfrom1to3mmapart,orahogbristlebrushworkswell. below 0.5%, because of borderline toxic effects on some
E1397–91 (2003)
hepatocyte cultures at DMSO concentrations of 1%. Both 3.4.2 All steps involving photographic emulsions should be
medium alone and solvent controls should be included in the done in total darkness. If absolutely necessary a red safelight
filter may be used sparingly.
experimental design. Concentrations of the test substance
3.4.3 Mount slides for each dose in plastic slide grips.
should be chosen that go just beyond cytotoxicity to about
Duplicate slides may be held in reserve.
1000-fold below the cytotoxic concentration. Cytotoxicity can
3.4.4 Mount a 50-mL disposable plastic beaker with tape
be determined by trypan blue dye exclusion or lactic dehydro-
into a slightly larger jar full of water. Place this assembly into
genase (LDH) release of the cultures or by morphological
a 42°C water bath and allow to reach the bath temperature.
examination of the fixed and stained cells at the end of the
3.4.5 KodakNTB-2 emulsionismostcommonlyused.The
experiment. Typical concentration ranges are from 10 to 0.001
emulsion may be used undiluted or can be diluted toa1to1
mM. Relatively insoluble substances should be tested up to
ratiowithdistilledwater.Iftheemulsionisdiluted,takecareto
their limit of solubility. Freely soluble, nontoxic chemicals
use double distilled or ultrapure water; thoroughly mix the
should not be tested at concentrations beyond 10 mM.Adose
solution but avoid formation of air bubbles. Undiluted emul-
of 10 mM dimethylnitrosamine (DMN) is required to produce
sion saves a step and provides slightly higher grain counts.
a strong DNA repair response in the assay. In contrast,
Meltemulsionina37°Cincubatorforatleast3h.Gentlypour
1,6-dinitropyrene induces DNArepair at concentrations as low
40to50mLoftheemulsionintothe50-mLdisposablebeaker.
as 0.00005 mM.
The unused portion can be resealed and stored under refrig-
3.3.3 Remove the WEI (see Annex A1) and replace with 2
eration. If one of the Ilford “K” series of photographic
mL of H-thymidine solution (see Annex A1) containing the
emulsions is used, it must not be liquefied and regelled.
dissolved test chemical. Place the cultures in the incubator for
3.4.6 Dip a test slide. Briefly turn on the red safelight and
16 to 24 h. During this period the compound may be metabo-
hold the slide up to it to make sure that there is enough
lized. If DNA damage occurs, it will be repaired, resulting in
emulsion in the cup to cover the cells, and that there are no
incorporation of the H-thymidine (see Annex A1).
bubbles in the emulsion.Air bubbles can be removed from the
3.3.4 Wash cultures twice with 4 mL WEI (see Annex A1)
surface of the emulsion by skimming the surface with a glass
per well.
slide. Turn off the safelight.
3.4.7 Dipeachgroupofslidesbyloweringthemintothecup
3.3.5 The remainder of these procedures are done with
until they touch the bottom. Pull the slides out of the emulsion
solutionsatroomtemperature.Replacethemediumwith4mL
with a smooth action to a 5-s count. Touch the bottom ends of
ofa1%sodiumcitratesolutionandallowtheculturestostand
the slides to a pad of paper towels to remove the bead of
for 10 min to swell the nuclei. The purpose for swelling the
emulsiononthebottom.Rememberthatallofthesestepsmust
cells is that the larger nuclei are more easily scored than the
take place in total darkness. Do not reuse the emulsion.
unswollen nuclei where the silver
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