ASTM E1397-91(2008)
(Practice)Standard Practice for In Vitro Rat Hepatocyte DNA Repair Assay (Withdrawn 2014)
Standard Practice for <i>In Vitro</i> Rat Hepatocyte DNA Repair Assay (Withdrawn 2014)
SIGNIFICANCE AND USE
Measurement of chemically induced DNA repair is a means of assessing the ability of a chemical to reach and alter the DNA. DNA repair is an enzymatic process that involves the recognition and excision of DNA-chemical adduct followed by DNA strand polymerization and ligation to restore the original primary structure of the DNA (7). This process can be quantitated by measuring the amount of labeled thymidine incorporated into the nuclear DNA of cells that are not in S-phase and is often called unscheduled DNA synthesis (UDS) (8). Numerous assays have been developed for the measurement of chemically induced DNA repair in various cell lines and primary cell cultures from both rodent and human origin (9). The primary rat hepatocyte DNA repair assay developed by Williams (10) has proven to be particularly valuable in assessing the genotoxic activity and potential carcinogenicity of chemicals (11), (12). Genotoxic activity is often produced by reactive metabolites of a chemical. The in vitro rat hepatocyte assay provides a system in which a metabolically competent cell is itself the target cell for measured genotoxicity. Most other short-term tests for genotoxicity employ a rat liver homogenate (S-9) for metabolic activation, which differs markedly in many important ways from the patterns of activation and detoxification that actually occur in hepatocytes. An extensive literature is available on the use of in vitro hepatocyte DNA repair assays (2, 3, 6, 13-28).
SCOPE
1.1 This practice covers a typical procedure and guidelines for conducting the rat in vitro hepatocyte DNA repair assay. The procedures presented here are based on similar protocols that have been shown to be reliable (1-6) .
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 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns 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.
WITHDRAWN RATIONALE
This practice covered a typical procedure and guidelines for conducting the rat in vitro hepatocyte DNA repair assay. The procedures presented were based on similar protocols that were shown to be reliable.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this practice was withdrawn in December 2013. This standard was withdrawn with no replacement because the assay is based on very old work and detects a very small subset of genotoxin and carcinogens.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E1397 − 91 (Reapproved2008)
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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope reactive metabolites of a chemical. The in vitro rat hepatocyte
assay provides a system in which a metabolically competent
1.1 This practice covers a typical procedure and guidelines
cell is itself the target cell for measured genotoxicity. Most
for conducting the rat in vitro hepatocyte DNA repair assay.
other short-term tests for genotoxicity employ a rat liver
The procedures presented here are based on similar protocols
2 homogenate (S-9) for metabolic activation, which differs
that have been shown to be reliable (1-6) .
markedly in many important ways from the patterns of
1.2 Mention of trade names or commercial products are
activationanddetoxificationthatactuallyoccurinhepatocytes.
meant only as examples and not as endorsements. Other
An extensive literature is available on the use of in vitro
suppliers or manufacturers of equivalent products are accept-
hepatocyte DNA repair assays (2, 3, 6, 13-28).
able.
1.3 The values stated in SI units are to be regarded as 3. Procedure
standard. No other units of measurement are included in this
3.1 Liver Perfusion:
standard.
3.1.1 All personnel must be knowledgeable in the proce-
1.4 This standard does not purport to address all of the
dures for safe handling and proper disposal of carcinogens,
safety concerns associated with its use. It is the responsibility
potential carcinogens, and radiochemicals. Disposable gloves
of the user of this standard to establish appropriate safety and
and lab coats must be worn.
health practices and determine the applicability of regulatory
3.1.2 Any proven technique which allows the successful
limitations prior to use.
isolation and culture of rat hepatocytes can be used. An
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
2.1 Measurement of chemically induced DNA repair is a
database is for male Fischer-344 rats.Young adult animals are
means of assessing the ability of a chemical to reach and alter
preferred. It is possible that factors such as sex, age, and strain
theDNA.DNArepairisanenzymaticprocessthatinvolvesthe
of the rat could affect the outcome of the DNA repair
recognitionandexcisionofDNA-chemicaladductfollowedby
experiments.Therefore,foranyoneseriesofexperimentsthese
DNAstrand polymerization and ligation to restore the original
variables (including controls) should be kept constant.
primary structure of the DNA (7). This process can be
3.1.4 Anesthetize the rat by intraperitoneal injection with a
quantitated by measuring the amount of labeled thymidine
50-g/mL solution of sodium phenabarbitol (0.2 mL per 100 g
incorporated into the nuclear DNA of cells that are not in
body weight) 10 min prior to the perfusion procedure. Other
S-phaseandisoftencalledunscheduledDNAsynthesis(UDS)
proven anesthetics are also acceptable. Make sure that the
(8). Numerous assays have been developed for the measure-
animal is completely anesthetized, so that it feels no pain.
ment of chemically induced DNA repair in various cell lines
3.1.5 Wet the abdomen thoroughly with 70% ethanol and
and primary cell cultures from both rodent and human origin
wipe with gauze for cleanliness to discourage loose fur from
(9).TheprimaryrathepatocyteDNArepairassaydevelopedby
getting on the liver when the animal is opened.
Williams (10) has proven to be particularly valuable in
3.1.6 Make a V-shaped incision through both skin and
assessing the genotoxic activity and potential carcinogenicity
muscle from the center of the lower abdomen to the lateral
ofchemicals (11), (12).Genotoxicactivityisoftenproducedby
aspects of the rib cage. Do not puncture the diaphragm or cut
theliver.Foldtheskinandattachedmusclebackoverthechest
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
to reveal the abdominal cavity.
Surgical Materials and Devices and is the direct responsibility of Subcommittee
3.1.7 Placeatubeapproximately1cmindiameterunderthe
F04.16 on Biocompatibility Test Methods.
Current edition approved Aug. 1, 2008. Published August 2008. Originally
back to make the portal vein more accessible.
approved in 1991. Last previous edition approved in 2003 as E1397–91 (2003).
3.1.8 Movetheintestinesgentlyouttotherighttorevealthe
DOI: 10.1520/E1397-91R08.
portal vein.Adjust the tube under the animal so that the portal
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this practice. vein is horizontal.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1397 − 91 (2008)
3.1.9 Put a suture in place (but not tightened) in the center 3.2.4 Gentlycomboutthecells,constantlyswirlingtheliver
of the portal vein and another around the vena cava just above while combing.Asterile metal dog-grooming comb with teeth
the right renal branch. spacedfrom1to3mmapart,orahogbristlebrushworkswell.
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 EGTAsolution (see AnnexA1)
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
3.1.11 Aperistalticpumpwithachangeablepumpheadand room temperature or heated to 37°C.
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
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
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(see AnnexA1) 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 (see AnnexA1) 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:
with4mLWEI(seeAnnexA1)perwelltoremoveunattached
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
E1397 − 91 (2008)
ethanol, should not exceed a 1% final concentration. Most 3.4.1 Any proven autoradiographic technique that yields
investigators try to limit the DMSO concentration to at or silver grains in proportion to the amount of incorporated
labeled thymidine may be used. Presented in 3.4.2-3.4.14 is a
below 0.5%, because of borderline toxic effects on some
typical procedure.
hepatocyte cultures at DMSO concentrations of 1%. Both
medium alone and solvent controls should be included in the 3.4.2 All steps involving photographic emulsions should be
done in total darkness. If absolutely necessary a red safelight
experimental design. Concentrations of the test substance
filter may be used sparingly.
should be chosen that go just beyond cytotoxicity to about
1000-fold below the cytotoxic concentration. Cytotoxicity can 3.4.3 Mount slides for each dose in plastic slide grips.
be determined by trypan blue dye exclusion or lactic dehydro- Duplicate slides may be held in reserve.
genase (LDH) release of the cultures or by morphological
3.4.4 Mount a 50-mL disposable plastic beaker with tape
examination of the fixed and stained cells at the end of the into a slightly larger jar full of water. Place this assembly into
experiment. Typical concentration ranges are from 10 to 0.001 a 42°C water bath and allow to reach the bath temperature.
mM. Relatively insoluble substances should be tested up to
3.4.5 KodakNTB-2 emulsionismostcommonlyused.The
their limit of solubility. Freely soluble, nontoxic chemicals
emulsion may be used undiluted or can be diluted toa1to1
should not be tested at concentrations beyond 10 mM.Adose
ratiowithdistilledwater.Iftheemulsionisdiluted,takecareto
of 10 mM dimethylnitrosamine (DMN) is required to produce
use double distilled or ultrapure water; thoroughly mix the
a strong DNA repair response in the assay. In contrast, solution but avoid formation of air bubbles. Undiluted emul-
1,6-dinitropyrene induces DNArepair at concentrations as low sion saves a step and provides slightly higher grain counts.
Meltemulsionina37°Cincubatorforatleast3h.Gentlypour
as 0.00005 mM.
40to50mLoftheemulsionintothe50-mLdisposablebeaker.
3.3.3 Remove the WEI (see Annex A1) and replace with 2
The unused portion can be resealed and stored under refrig-
mL of H-thymidine solution (see Annex A1) containing the
eration. If one of the Ilford “K” series of photographic
dissolved test chemical. Place the cultures in the incubator for
emulsions is used, it must not be liquefied and regelled.
16 to 24 h. During this period the compound may be metabo-
3.4.6 Dip a test slide. Briefly turn on the red safelight and
lized. If DNA damage occurs, it will be repaired, resulting in
hold the slide up to it to make sure that there is enough
incorporation of the H-thymidine (see Annex A1).
emulsion in the cup to cover the cells, and that there are no
3.3.4 Wash cultures twice with 4 mL WEI (see Annex A1)
bubbles in the emulsion.Air bubbles can be removed from the
per well.
surface of the emulsion by skimming the surface with a glass
3.3.5 The remainder of these procedures are done with
slide. Turn off the safelight.
solutionsatroomtemperature.Replacethemediumwith4mL
3.4.7 Dipeachgroupofslidesbyloweringthemintothecup
ofa1%sodiumcitratesolutionandallowtheculturestostand
until they touch the bottom. Pull the slides out of the emulsion
for 10 min to swell the nuclei. The purpose
...
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