ASTM E1398-91(2008)
(Practice)Standard Practice for In Vivo Rat Hepatocyte DNA Repair Assay (Withdrawn 2014)
Standard Practice for <i>In Vivo</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 Rat Hepatocyte DNA Repair Assay (Withdrawn 2014)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 recognition and excision of DNA-chemical adducts, followed by DNA strand polymerization and ligation to restore the original primary structure of the DNA (6). 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) (7). 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 (4). The primary culture rat hepatocyte DNA repair assay has proven to be particularly valuable in assessing the genotoxic activity of chemicals (8). Genotoxic activity often results from metabolites of a chemical. The in vitro rat hepatocyte assay provides a system in which a metabolically competent cell is also the target cell. Most other in vitro 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 DNA repair assays (9-19).
A further advance was the development of an in vivo rat hepatocyte DNA repair assay in which the test chemical is administered to the animal and the resulting DNA repair is assessed in hepatocytes isolated from the treated animal (20). Numerous systems now exist to measure chemically induced DNA repair in specific tissues in the whole animal (4). The average of in vivo assays is that they reflect the complex patterns of uptake, distribution, metabolism, detoxification, and excretion that occur in the whole animal. Further, factors such as chronic exposure, sex differences, and different routes of exposure can be studied ...
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 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 vivo hepatocyte DNA repair assay.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this practice was withdrawn in December 2013. This standard has been 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: E1398 − 91(Reapproved 2008)
Standard Practice for
In Vivo Rat Hepatocyte DNA Repair Assay
This standard is issued under the fixed designation E1398; 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 provides a system in which a metabolically competent cell is
also the target cell. Most other in vitro short-term tests for
1.1 This practice covers a typical procedure and guidelines
genotoxicityemployaratliverhomogenate(S-9)formetabolic
forconductingtheratinvivohepatocyteDNArepairassay.The
activation, which differs markedly in many important ways
procedures presented here are based on similar protocols that
2 from the patterns of activation and detoxification that actually
have been shown to be reliable (1, 2, 3, 4, 5).
occurinhepatocytes.Anextensiveliteratureisavailableonthe
1.2 Mention of trade names or commercial products are
use of in vitro DNA repair assays (9-19).
meant only as examples and not as endorsements. Other
2.2 Afurther advance was the development of an in vivo rat
suppliers or manufacturers of equivalent products are accept-
hepatocyte DNA repair assay in which the test chemical is
able.
administered to the animal and the resulting DNA repair is
1.3 The values stated in SI units are to be regarded as
assessed in hepatocytes isolated from the treated animal (20).
standard. No other units of measurement are included in this
Numerous systems now exist to measure chemically induced
standard.
DNA repair in specific tissues in the whole animal (4). The
1.4 This standard does not purport to address all of the
average of in vivo assays is that they reflect the complex
safety concerns associated with its use. It is the responsibility
patterns of uptake, distribution, metabolism, detoxification,
of the user of this standard to establish appropriate safety and
and excretion that occur in the whole animal. Further, factors
health practices and determine the applicability of regulatory
such as chronic exposure, sex differences, and different routes
limitations prior to use.
of exposure can be studied with these systems. This is
illustrated by the potent hepatocarcinogen 2,6-dinitrotoluene
2. Significance and Use
(DNT). Metabolic activation of 2,6-DNT involves uptake,
2.1 Measurement of chemically induced DNA repair is a
metabolism by the liver, excretion into the bile, reduction of
means of assessing the ability of a chemical to reach and alter
the nitro group by gut flora, readsorption, and further metabo-
the DNA. DNA repair is an enzymatic process that involves
lism by the liver once again to finally produce the ultimate
recognition and excision of DNA-chemical adducts, followed
genotoxicant (21). Thus, 2,6-DNT is negative in the in vitro
by DNA strand polymerization and ligation to restore the
hepatocyte DNArepair assay (22) but is a very potent inducer
original primary structure of the DNA (6).This process can be
of DNA repair in the in vivo DNA repair assay (23, 24).A
quantitated by measuring the amount of labeled thymidine
problem with tissue-specific assays is that they may fail to
incorporated into the nuclear DNA of cells that are not in
detect activity of compounds that produce tumors in other
S-phaseandisoftencalledunscheduledDNAsynthesis(UDS)
target tissues. For example, no activity is seen in the in vivo
(7). Numerous assays have been developed for the measure-
DNA repair assay with the potent mutagen benzo(a)pyrene
ment of chemically induced DNA repair in various cell lines
(BP), probably because limited tissue distribution and greater
and primary cell cultures from both rodent and human origin
detoxification in the liver yields too few DNA adducts to
(4). The primary culture rat hepatocyte DNA repair assay has
produce a measurable response (3). In contrast, BP is readily
proven to be particularly valuable in assessing the genotoxic
detected in the less tissue-specific in vitro hepatocyte DNA
activity of chemicals (8). Genotoxic activity often results from
repair assay (11). An extensive literature exists on the use of
metabolites of a chemical. The in vitro rat hepatocyte assay
the in vivo hepatocyte DNA repair assay (1-3, 5, 9, 25-33).
1 3. Procedure
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devices and is the direct responsibility of Subcommittee
3.1 Treatment:
F04.16 on Biocompatibility Test Methods.
Current edition approved Aug. 1, 2008. Published August 2008. Originally
3.1.1 All personnel must be knowledgeable in the proce-
approved in 1991. Last previous edition approved in 2003 as E1398–91 (2003).
dures for safe handling and proper disposal of carcinogens,
DOI: 10.1520/E1398-91R08.
potential carcinogens, and radiochemicals. Disposable gloves
The boldface numbers in parentheses refer to the list of references found at the
end of this practice. and lab coats must be worn.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1398 − 91 (2008)
3.1.2 Any strain or sex of rat may be used. The largest 3.2 Liver Perfusion:
database is for male Fischer-344 rats.Young adult animals are
3.2.1 Any proven technique which allows the successful
preferred. It is possible that factors such as sex, age, and strain
isolation and culture of rat hepatocytes can be used. An
of the rat could affect the outcome of the DNA repair
example of one such procedure is given in 3.2.2-3.2.17.
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-
3.1.3 Administration is usually by gavage with chemicals
surable for at least 16 h after treatment. More commonly,
dissolved or suspended in an appropriate vehicle such as water
however, chemicals (for example, 2,6-DNT and
or corn oil, depending on solubility.An advantage of the assay
2-acetylaminofluorene (2-AAF)) show a peak response 12 to
is that various routes of administration may be chosen. Thus,
16 h post-treatment.The time from treatment to perfusion may
chemicals may also be administered by intraperitoneal injec-
bevariedtoobtainatimecourseofinducedrepair.Theroutine
tion or inhalation or in the diet. For gavage administration, 0.2
protocol for primary screening involves a time point between
to 1.0 mL of test chemical solution is administered per 100 g
12 and 16 h with an optional time point between 1 and 4 h.
bodyweight.Controlsreceivetheappropriatevehiclesolution.
3.2.3 Anesthetize the rat by intraperitoneal injection with a
Stock corn oil should be replaced with fresh monthly.
50-mg/mLsolution of sodium pentobarbital (0.2 mLper 100 g
3.1.4 ForDNArepairstudies,animalsmaybetakenofffeed
body weight) 10 min prior to the perfusion procedure. Other
for a few hours prior to sacrifice to make the process of
proven anesthetics are also acceptable. Make sure that the
perfusion a little easier with less food in the stomach. The
animal is completely anesthetized, so that it feels no pain.
period without food should never exceed 12 h because of the
3.2.4 Secure the animal with the ventral surface up on
possibility of altered metabolism or uptake. Water should be
absorbent paper attached to a cork board. Fold the paper in on
continuously available.
each edge to contain perfusate overflow. Thoroughly wet the
3.1.5 Dose selection will depend on the characteristics of
abdomen with 70% ethanol and wipe with gauze for cleanli-
each chemical and the purpose of the experiment. If one is nessandtodiscourageloosefurfromgettingontheliverwhen
investigating whether a chemical can produce a genotoxic
the animal is opened.
effect in the animal, even at massive doses and by routes of
3.2.5 Make a V-shaped incision through both skin and
administration that may overwhelm natural defense
muscle from the center of the lower abdomen to the lateral
mechanisms,thenhighdoses(suchastheLD50,orhigher)that
aspects of the rib cage. Do not puncture the diaphragm or cut
do not kill the animal before the 16-h sacrifice point may be
theliver.Foldbacktheskinandattachedmuscleoverthechest
employed. Even in such a case, doses above 1000-mg/kg body
to reveal the abdominal cavity.
weightarenotrecommended.Insomeinstanceshepatotoxicity
3.2.6 Placeatubeapproximately1cmindiameterunderthe
at high doses may result in inhibition of cell attachment or
back to make the portal vein more accessible.
DNA repair. More commonly, the purpose of employing the
3.2.7 Movetheintestinesgentlyouttotherighttorevealthe
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×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
mayswitchfromtheEGTAsolutiontothecollagenasesolution
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.Ifapositiveresponseisseen,additionalexperimentsare
portal vein.
conducted to establish the dose-response relationship. If no
3.2.9.1 Aperistalticpumpwithachargeablepumpheadand
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
datapoint.
3.2.9.2 Begin the flow of the 37°C EGTA solution at 8
mL/min and run to waste.
3.1.6 Thus far, no examples have been seen of a compound
thatproducesaUDSresponseinfemaleratsbutnotmales.For
3.2.10 Cannulate the portal vein with a 20 GA 1 ⁄4-in.
thosecaseswhereacomparisonhasbeenmade,malesrespond catheter about 3 mm below the suture. Remove the inner
more strongly than females in this assay.Thus, for the purpose
needle and insert the plastic catheter further to about ⁄3 the
of routine screening only male rats need to be used. length of vein and tie in place by the suture. Blood should
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
exposure to expired chemicals. Contaminated cages, bedding, 3.2.11 Immediately cut the vena cava below the right renal
excreta, and carcasses should be disposed of safely according branch and allow the liver to drain of blood for 1.5 min. The
to standard published procedures. liver should rapidly clear of blood and turn a tan color. If all
E1398 − 91 (2008)
lobes do not clear uniformly, the catheter has probably been 3.3.8.1 Some laboratories prefer to keep the cells on ice
inserted too far into the portal vein. until ready for use, while others keep them at room tempera-
ture. Cells should be used as soon as possible, preferably
3.2.12 Tightenthesuturearoundthevenacavaandincrease
within 1 h.
the flow to 20 mL/min for 2 min.The liver should swell at this
3.3.9 Determine viability and cell concentration by the
point. In some cases gentle massaging of the liver or adjusting
method of trypan blue exclusion. The preparation should be
the orientation of the catheter may be necessary for complete
primarilyasingle-cellsuspensionwithaviabilityofover60%
clearing. At this point the vena cava above the suture may be
for control cultures. With practice and the proper technique,
clipped to release some of the pressure in the liver.
viabilitiesofabout90%canroutinelybeobtained.Attachment
3.2.13 Switch the flow to the 37°C collagenase solution for
of the cells to the substrate is an active process. Thus, if a
12 min. During this period, cover the liver with sterile gauze
sufficientnumberofcellsattachtoconducttheexperiment,this
wetted with sterile saline or WEI (see Annex A1) and place a
is a further indication of the viability of the culture.
40-W lamp 2 in. above the liver for warming. It is valuable to
3.3.10 Placea25-mmroundplasticcoverslipintoeachwell
screen each new batch of collagenase to be ensured that it will
of 6-well culture dishes. 10.5 by 22-mm plastic coverslips and
function properly.
26 by 33-mm eight-chamber culture dishes can also be used.
3.2.14 Allowtheperfusatetoflowontothepaperandcollect
Be sure to keep the proper side up as marked on the package.
by suction into a vessel connected by means of a trap to the
FourmillilitresofWEC(seeAnnexA1)areaddedtoeachwell.
vacuumline.Theperfusateshouldbedisposedofashazardous
Hepatocyteswillnotattachtoglassunlesstheslideshavebeen
waste.
boiled. The use of collagen-coated thermanox coverslips im-
3.2.15 Aftertheperfusioniscompleted,removethecatheter
proves cell attachment and morphology.
and gauze. Remove the liver carefully by cutting away the
3.3.10.1 Theseproceduresyieldpreparationsconsistingpri-
membranes connecting it to the stomach and lower esophagus,
marily of hepatocytes.Approximately 400000 viable cells are
cutting away the diaphragm, and cutting any remaining attach-
seeded into each well and distributed over the coverslip by
ments to veins or tissues in the abdomen.
shaking or stirring gently with a plastic 1-mL pipet. Glass
3.2.16 Hold the liver by the small piece of attached dia-
pipettes can scratch the coverslips.
phragm and rinse with sterile saline or WEI (see Annex A1).
3.3.11 Incubate the cultures for 90 to 120 min in a 37°C
3.2.17 Place the liver in a sterile petri dish and take to a
incubator with 5% CO and 95% relative humidity to allow
sterile hood to prepare the cells.
the cells to attach.
3.3 Preparation of Hepatocyte Cultures:
3.4 Labeling the Cultures:
3.4.1 After the 90-min attachment, wash cultures once with
3.3.1 Place the perfused liver in a 60-mm p
...
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