grain counting in the in vitro hepatocyte dna-repair assay

Mutation Research, 272 (1992) 9-15 9 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1161/92/$05.00

MUTENV 08824

Grain counting in the in vitro hepatocyte DNA-repair assay

Giovanni Brambilla and Antonietta Martelli Institute of Pharrnacology, University of Genoa, 1-16132 Genoa (Italy)

(Received 31 December 1991) (Accepted 7 January 1992)

Keywords: Hepatocyte DNA-repair assay; Unscheduled DNA synthesis, autoradiographic evaluation

Summary

The in vitro hepatocyte DNA-repair assay is a widely used useful method in assessing the genotoxic activity of both directly and indirectly acting chemical agents. This article discusses the criteria presently employed in the autoradiographic evaluation of unscheduled DNA synthesis, and suggests that the subtraction of either the average or the highest cytoplasmic grain count, usually carried out to obtain the net nuclear grain count, may represent a potential source of errors when the test compound is a weakly genotoxic or a non-genotoxic agent. As a matter of fact, a response can be classified as positive or negative depending on the procedure used to quantitate the cytoplasmic background, and the subtraction of this background from the nuclear count is not founded on a sound theoretical basis because of the following reasons: the different nature of the processes responsible for the generation of nuclear and cytoplasmic grains; and the quantitatively different effect that the test compounds may have on the nuclear and the cytosolic labelling.

The autoradiographic detection of unscheduled DNA synthesis (UDS) in rat hepatocyte primary cultures (HPC) is widely used to measure excision repair of DNA lesions induced by both directly and indirectly acting genotoxic agents. In the last few years some papers have been pub- lished to provide a typical procedure and guidelines for conducting the in vitro rat hepatocyte DNA-repair assay (Butterworth et al., 1987; Har- bach et al., 1989, 1991; Hill et al., 1989; Swierenga et al., 1991). The purpose of this short article is to

Correspondence: Dr. G. Brambilla, Istituto di Farmacologia dell'Universit~t, Viale Benedetto XV, 2, 1-16132 Genoa (Italy).

discuss the criteria proposed for the evaluation of the biological end point of this assay, which corresponds to the repair synthesis of chromosomal DNA in the nucleus.

Experimentally UDS is measured by counting the silver grains produced in the layer of photo- graphic emulsion covering the nucleus by the decay of [3H]thymidine that has been incorporated into the nuclear DNA of non-S-phase hepatocytes. A serious technical problem in measuring UDS is the presence of variable cytoplasmic labelling, which is referred to as cytoplasmic background. The cytoplasmic grains are mostly due to incorporation of [3H]thymidine into mitochondrial DNA. Considering that a certain proportion of the nuclear grain count can be attributed to

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[3H]thymidine incorporation into the layer of cytoplasm placed between the nucleus and the autoradiographic emulsion, the nuclear count is cor- rected by subtracting an appropriate cytoplasmic grain count; this results in a value referred to as net nuclear grains (NG). The cytoplasmic count corresponds to the number of grains located above an area of cytoplasm that has the same size as the nucleus and is directly adjacent to it; however, to cope with the irregular distribution of grains, various methods have been developed for obtain- ing this value. In brief, as cytoplasmic counts have been used: the average of 3 cytoplasmic counts, the highest of 3 cytoplasmic counts, the average of 2 cytoplasmic counts, the highest value of 2 counts carried out over the 2 most heavily labelled cytoplasmic areas (subjectively chosen), and the count of 1 cytoplasmic area without mention as to how this area was selected. Recently Har- bach et al. (1991) compared the results obtained using 4 scoring methods, and concluded that subtracting the average, instead of the highest cytoplasmic count, merely shifts the NG values higher by about 3 -6 grains with essentially no change in the statistical significance of the results.

In the case of control cells there are usually more grains per unit area in the cytoplasm than in the nucleus so that the NG is often a negative number. The reason proposed to explain why the control NG value tends to be less than zero is that the cytoplasm is thinner over the nucleus than over the rest of the cell.

The criteria presently used for the acceptabil- ity of the UDS assay and for the definition of a positive or negative response are based on the above-mentioned concepts and the consequent procedure for the evaluation of NG values. There is substantial general agreement on these criteria, and they can be summarized as follows.

(1) Using the recommended experimental conditions, NG values of negative controls (untreated a n d / o r solvent) should be lower than zero. For instance, average control values ranging from -11 .1 to - 2 9 . 9 are reported by Williams et al. (1989), and Butterworth et al. (1987) indicate an expected range from - 1 0 to - 2 , with 0 -10% repairing cells. Harbach et al. (1989) state that the UDS assay is considered acceptable only if the solvent control group yields _< 0 NG.

(2) According to the most commonly used criterion, a test compound is considered positive if the NG of any tested concentration is >_ 5, and if the percentage of cells in repair is > 10% (or > 20%). Other criteria are the existence of a positive dose- response relationship, and a statis- tically significant increase over controls (Williams et al., 1989). If the highest NG count is between (/ and 5 the results are considered either potentially weakly positive or inconclusive.

(3) The results are considered negative if all the concentrations tested have a NG count of _< 0. However, negative results are accepted only if the chemical used as positive control yielded NG counts in the usual range.

In our opinion there are at least three reasons which make questionable this way of evaluating NG; the first is presumably of a technical nature, whilst the other two, already raised by Lonati- Galligani et al. (1983) and by Rossberger and Andrae (1987) but not taken into account, are of theoretical importance and based on experimental evidence.

The first reason concerns the difference between the average NG counts of untreated or solvent-treated controls reported in papers pub- lished in the years from 1977 to 1981 and those appearing after 1989. To make possible a comparison we report a few representative examples in Table 1; the experiments chosen were carried out in substantially similar conditions, and for each of them the method used to evaluate the cytoplasmic background is indicated. The data listed show that average NG counts usually ranged from - 2.0 to + 1.0 in the first years, and in contrast reached values often below - 10.0 and sometimes near to or even lower than -20 .0 in recent years. Since it is unreasonable to attribute this event to in- creased [3H]thymidine incorporation into the cytoplasm, i.e., to a true increase of cytoplasmic background, a possible cause might be the criterion employed to quantify the cytoplasmic background. This is suggested by a comparison of NG values obtained by subtracting the highest or the average cytoplasmic background, and in this respect, the results obtained by Harbach et al. (1991), who compared 4 methods of scoring cytoplasmic grains, can be considered illustrative. Their Table 4 shows that for the same experiment

the average NG counts of DMSO-treated cultures varied from -15 .7 to -0 .9 ; the highest value was obtained by subtracting from the nuclear grain count the maximum cytoplasmic count of the 2 most heavily labelled cytoplasmic areas adjacent to the nucleus (30.4); the lowest when the value subtracted was the average count of 2 randomly selected cytoplasmic areas (19.0). With a compound producing at the most effective dose a gross nuclear count of 25.0, assuming the same level of cytoplasmic background in treated cells, the NG obtained with the first criterion would have been -5 .4 , and the NG obtained with the second criterion +6.0. Thus the response can be classified as positive or negative depending on the criterion used to evaluate the cytoplasmic background. In this respect, it is worth noting that in the same Table 4 the NG value of - 5 . 8 produced by exposure to 50 nM 2-acetylamino- fluorene, although significantly different (p < 0.05) from the corresponding control value (NG

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= - 15.7), cannot be considered a positive result, being < 0.

Another possible if not likely reason for very negative NG counts might be a high emulsion background, i.e., a background formed by silver grains not due to radiation from the experimental source but to other causes. These various causes have been illustrated by Rogers (1973), and it is known that the emulsion background can have an uneven distribution. Moreover, the emulsion background might be lower above the nucleus than above the cytoplasm because of a reduced thickness of the emulsion in the former site, and this could contribute to a further reduction of NG counts.

Finally, it should be considered that grain counting is generally accomplished with an automated system that measures the area of the grain. A conversion factor calculated as [number of grains counted manual ly /measured area of the same grains] is used to convert the machine counts

TABLE 1

OLD AND RECENT VALUES OF NET NUCLEAR GRAIN COUNT IN UNTREATED OR SOLVENT-TREATED RAT HPC

Method for Number scoring of cytoplasmic experi- grains a ments

Net nuclear grain count b

Mean -+ SD Range

Reference

Av. 3 a. 1 0.8 Williams, 1977 Hi. 3 a. 4 - 1.2 _+ 1.4 - 2.3/ 0.8 Williams, 1978 Hi. 3 a. 1 -3 .2 Bermudez et al., 1979 Av. 3 a. 7 1.3 ___ 0.4 0 .7/ 2.0 Probst and Neal, 1980 Av. 3 a. 1 1.1 Probst et al., 1980 Hi. 3 a. 1 0 Williams et al., 1981 Av. 3 a. 91 - 0 . 2 + 0 . 6 - 2 . 0 / 1.1 Probst et al., 1981 Av. 3 a. 40 - 1.7 -+ 0.6 - 0 . 3 / 3.0 Hill et al., 1989 Hi. 3 a. 16 - 2 9 . 9 / - 11.1 Williams et al., 1989 Av. 2 a. (?) 21 -11.7-+3.3 - 2 0 . 1 / - 3 . 8 Harbach et al., 1989 Hi. 2 h.a. 3 - 14.2-+5.9 - 19 .2 / -7 .6 Harbach et al., 1991 Av. 2 h.a. 3 - 10.2_+5.8 - 1 4 . 9 / - 3 . 7 Hi. 2 r.a. 3 -6 .1 -+2.9 - 9 . 4 / - 4 . 3 Av. 2 r.a. 3 -2.1-+2.2 - 4 . 6 / - 0 . 8

a Methods used for selecting the nuclear-sized cytoplasmic background to be subtracted from the nuclear count in order to calculate the net nuclear grain count: Av. 3 a., average of 3 areas; Hi. 3 a., highest of 3 areas; Av. 2 a., average of 2 areas; Hi. 2 h.a., highest of the 2 most heavily labelled areas; Av. 2 h.a. , average of the 2 most heavely labelled areas; Hi. 2 r.a., highest of 2 randomly selected areas; Av. 2 r.a., average of 2 randomly selected areas. When more than one datum was available, the values listed are the mean a n d / o r the range of average net nuclear grains of control HPC reported in the corresponding article.

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to actual grains. It cannot be excluded that this procedure is a source of error, in particular when the emulsion background is high, or when the sensitivity of the counting system is not adjusted so that only silver grains are counted. In this respect it is worth noting that the guidelines of the UKEMS subcommittee for in vivo rat liver UDS assay (personal communication of Dr. J.C. Kennelly) suggest that 'deep staining of structures such as the nucleolus is to be avoided as an image analyzer may score them along with emulsion silver grains', and admit that 'consequently, in most UDS autoradiographs, the essential delin- eation of the cytoplasm is difficult to record pho- tographically'.

In the UDS assays on rat hepatocytes carried out in our laboratory silver grains have always been counted manually because of the absence of an automated counting system; autoradiographs with a high emulsion background have been always discarded; and the criterion usually followed to obtain N G was the subtraction from the nuclear count of the cytoplasmic background present in a nuclear-sized randomly chosen cytoplasmic area adjacent to the nucleus. In these conditions, in a series of 41 experiments on duplicate cultures from 41 rats the average NG values of untreated controls were between - 1 . 3 and 0 in 22% of experiments, between 0 and + 1.0 in 56%, and between + 1.0 and +2.7 in the remaining ones. The distribution of nuclear grain counts and of cytoplasmic grain counts in 500 untreated rat hepatocytes is illustrated in Fig. 1. Fig. 2 shows the histogram of NG distribution in 1000 control hepatocytes and in 1000 hepatocytes exposed to the hepatocarcinogen N-nitrosodimethylamine; a similar histogram of 400 control hepatocytes and 300 hepatocytes exposed to 2-chloroethanol, which has been found to be non-carcinogenic in mice and rats of both sexes (Tennant et al., 1987), is shown in Fig. 3. NG counts of individual untreated hepatocytes, which are reported in the upper section of Figs. 2 and 3, show a normal distribution, which suggests that an individual cell may be assumed to be in repair (p < 0.05) if it contains > 7 net grains. The lower section of Fig. 2 shows the strong UDS response elicited by a 5 mM concentration of N-nitrosodimethylamine (NDMA). The lower section of

NUCLEAR GRAINS

CYTOPLASMIC GRAINS

I I l l l l l I I l l l I I l l l 1 5 10 15

Fig. I. Distribution of nuclear grain counts and of cytoplasmic grain counts in 500 untreated rat hepatocytes obtained from 5

different rats.

Fig. 3 shows the negative UDS response obtained with the maximal subtoxic concentration (0.5 mM) of 2-chloroethanol.

Reasons based on both theoretical grounds and experimental evidence that make questionable the criteria used at present to calculate NG were first discussed by Lonati-Galligani et al. in 1983. These reasons can be summarized as follows. Taking into account that the cytoplasmic

CONI'IROL HE~ATOCYTES

5 mM NDMA-TREATED HEPATOCYTES

I ~ l l l l l l l ~ l ~ l l l l l ~ l l l l l -10 0 10 20 30 40 50 60 70 80 90 100

NG VAU.ES

Fig. 2. Histograms of the distribution of NG values in 1000 controls and in 1000 N-nitrosodimethylamine (5 mM)-treated hepatocytes obtained from 10 male Sprague-Dawley rats. Concerning the control population, the mode was 0, the mean 0.59, the SEM 0.11 and the SD 3.61; therefore 95% of the control population lay between - 6 and 9 net grains. In the treated population, the mode was 10, the mean 23.67, the SEM 0.60 and the SD 10.06; therefore 95% of the treated

population lay between 6 and 69 net grains.

layer cover ing the nucleus is subs tant ia l ly th inner than the layer immed ia t e ly next to it, the p roce- du re of sub t rac t ing f rom the gross nuc lea r count the cy toplasmic b a c k g r o u n d is a rb i t ra ry and p rob- ably resul ts in a var iab le overcor rec t ion . F u r t h e r - more , the cy toplasmic labe l l ing is due mainly to i nco rpo ra t ion of [3H]thymidine into mi tochon- dr ia l D N A ; the re fo re it may be in f luenced by the c o m p o u n d tes ted , and in fact it was found to be

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marked ly r e d u c e d by e t h i d i u m b r o m i d e and sig- nif icant ly inc reased by th iourea . As a consequence, when only N G counts a re cons idered , fa lse-posi t ive resul ts may be g e n e r a t e d by a s t rong r educ t ion of cy toplasmic label l ing, and false- negat ive resul ts by an e n h a n c e d cy top lasmic background . In view of this s i tua t ion , L o n a t i - G a l - l igani et al. sugges ted tha t to d raw a conclus ion with r ega rd to the induc t ion of U D S a d o s e - r e - sponse curve should be cons t ruc ted for the nu- c lear as well as for the cy toplasmic label l ing and the t r end of bo th inco rpora t ions should be con- s idered. F u r t h e r examples of the diff icul t ies and pi tfal ls resul t ing f rom the effect of the test com- p o u n d on cytosolic label l ing have been r e p o r t e d by Rossbe rge r and A n d r a e (1987) and can be drawn from the resul ts o b t a i n e d with N-n i t roso- d ipheny lamine (Fau t z et al., 1991).

,, Ill I ' 1

II

CONTROL HEPATOCYTES

I I J " ' " '

0.5 mM CHLOROETHANOL-TREATED HEPATOCYTES

I I I I I I -10 -5 0 ÷5 +10 +15 *20

NG VALUES

Fig. 3. Histograms of the distribution of NO values in 400 controls and in 300 2-chloroethanol (0.5 mM)-treated hepatocytes obtained from 4 male Sprague-Dawley rats. Concerning the control population, the mode was 1, the mean 0.25, the SEM 0.15 and the SD 3.01; therefore 95% of the control population lay between - 6 and 5 net grains. In the treated population, the mode was 4, the mean 2.93, the SEM 0.23 and the SD 3.97; therefore 95% of the treated population lay

between - 3 and 10 net grains.

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Even more important for a correct evaluation of UDS are the findings of R o s s b e r g ~ and An- drae (1987) who investigated the causes of the cytosolic and the nuclear labelling in autoradiographs of control rat HPC, and proved that the former is mainly due to mitochondrial DNA synthesis and the latter essentially reflects unscheduled synthesis of chromosomal DNA. By density- gradient centrifugation of D N A labelled with BrdUrd and [3H]deoxycytidine they showed that considerable DNA-repa i r synthesis occurs in rat hepatocyte nuclei even in the absence of an added gen6toxic agent, and demonstrated that this event is the consequence of D N A breaks generated in chromosomal D N A by both the bacterial collagenase used in the cell isolation and the decay of the radioactive D N A precursor used in the mea- surement of UDS. A comparison by autoradiographic analysis of the distribution of silver grains over the nucleus in intact non-S-phase cells and in isolated nuclei confirmed that the vast majority of radioactivity was incorporated into the nucleus and not into the overlying cytoplasm or mitochondria. Moreover, the staining of mitochondria with the specific dye rhodamine 123 showed that very few of them are actually located over the nucleus. With respect to collagenase, it can be added that Cesarone et al. (1984) demonstrated in hepatocytes obtained by liver perfusion with this enzyme the presence of a small but significant amount of D N A damage as evaluated by the alkaline elution technique. This DNA-damaging activity of collagenase is confirmed by the observation that in suspensions of hepatocytes from untreated rats prepared with this enzyme the frequency of D N A lesions, when measured by a very sensitive viscometric technique (Brambilla et al., 1987), was markedly higher than that detected in suspensions obtained without the use of collagenase, and sufficient to preclude the detection of DNA breaks produced by a weakly active genotoxic agent (Brambilla, unpublished results).

In conclusion, both strong and less strong reasons suggest that the practice at present generally employed and substantially imposed for the autoradiographic evaluation of D N A repair in rat HPC, i.e., that of subtracting the cytoplasmic grains from nuclear grains and expressing UDS as net grains per nucleus, is not founded on a sound

theoretical basis and may represent a potential source of error when the test compound is a weakly genotoxic or a non-genotoxic agent.

The strongest reason consists in the different nature of the two processes responsible for the generation of nuclear and cytoplasmic grains: the former are due, even in untreated cells, to the repair of chromosomal DNA lesions; the latter reflect DNA synthesis in mitochondria or radioactivity bound to proteins.

Another strong reason is related to the quantitatively and qualitatively different effects that the test compound may exert on the nuclear and the cytoplasmic labelling.

The third, perhaps weaker, reason concerns the fact that at present the criterion most widely employed to evaluate NG is that of subtracting the highest possible cytoplasmic background, without taking into account that, if even a fraction of the grains above the nucleus is due to cytoplasmic labelling, this fraction is undoubtedly lower than the cytoplasmic background of a nuclear sized area of the adjacent cytoplasm. Be- sides the possibility of giving rise to false-negative results, this criterion can produce the not negligi- ble consequence that an UDS assay could be considered not acceptable simply because the NG value of the solvent control group is > 0. Last year we twice had a negative comment from two referees who judged that our experiments should have been rejected due to the positive values (0.5-1.0) obtained in the solvent controls.

Because of all these reasons we agree with the suggestion of Lonati-Galligani et al. (1983), which is shared by Rossberger and Andrae (1987), that for a correct evaluation of UDS it is convenient to monitor both cytoplasmic and nuclear grains and present them separately. Net nuclear grains can represent further useful information but, instead of the highest, the average or perhaps bet- ter the lowest cytoplasmic labelling should be subtracted. In the absence of an effect of the test compound on cytoplasmic labelling, a satisfactory criterion for a positive response might be an increase in the mean NG count that exceeds 2 SDs of the concurrent control population. Be- cause of the possibility that NG values are influ- enced by experimental conditions, in each individual study the mean NG counts of treated

hepatocytes should be compared with the mean NG counts of the concurrent control, without reference to historical control values. Taking into account that results obtained in the past and our experience clearly indicate that mean NG counts of negative controls may be higher than zero, the statement that the UDS assay is considered acceptable only if the control group yields < 0 NG is arbitrary.

Acknowledgements

The authors wish to thank Drs. C.M. Williams and J.C. Kennelly for their valuable comments on the manuscript.

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grain counting in the in vitro hepatocyte dna-repair assay

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