PLEASE NOTE THAT TO OBTAIN DETAILS OF THE PROTOCOL YOU SHOULD REGISTER

TO OBTAIN DETAILED PROTOCOL YOU SHOULD FIRST REGISTER AT ECVAM SIS

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

Protocol no. 48
LUNG CELL ASSAY

Potential embryotoxicity is assessed by monitoring the effect of the test compound on total protein synthesis, and DNA synthesis in cultured human foetal lung fibroblasts. Rat lung epithelial cells can be used to determine cytotoxicity of select compounds because of their ability to metabolise xenobiotics.

CONTACT

Dr. Frank A. Barile
Department of Natural Sciences
York College
The City University of New York Jamaica
New York 11451 USA
Tel: USA - 718 262 2720 Fax: USA - 718 262 2027

RATIONALE

The effect of a substance on cultured human foetal lung fibroblasts can be taken as a measure of its embryotoxic potential. Further information may be obtained by using immortal rat lung epithelial cell cultures which retain a capacity to metabolize xenobiotics.

BASIC PROCEDURE

Lung cells are cultured for 3 or 7 days, or until confluency is reached in the absence or presence of the test substance. Total protein synthesis is determined by measuring the incorporation of 3H-proline into trichloroacetic acid-insoluble material. DNA synthesis is measured by the uptake of tritiated thymidine into the trichloroacetic acid-insoluble fraction.

CRITICAL ASSESSMENT

Investigations of teratogenic and embryotoxic potential have traditionally relied on whole animal models. However, such models introduce a large and undefinable level of variability, since any toxic physical and biochemical effects will be influenced by variations in levels of hormones and growth factors, and in the absorption, distribution and elimination of the test substance. In vitro systems, on the other hand, are independent of nutritional and hormonal influence and can therefore elucidate the cellular basis of toxicity. The human foetal lung fibroblasts used in this test system (HFL1) have been extensively characterised (Breul et al., 1980, Hayflick, 1965, Jacobs et al., 1976) and are suitable for screening chemicals with potential toxicity to foetal development. They derive from a first-trimester foetus, and thus represent the gestational stage when the foetus is most susceptible to developmental anomalies. Other lung cells, such as Clara and epithelial cells, have the advantage that they contain enzymes capable of metabolising xenobiotics (Devereux et al., 1982). However, long-term culture of these cells from mammalian embryos has yet to be achieved. Moreover, differentiated alveolar epithelium arises at a much later stage of foetal development which is less susceptible to toxic processes. Nevertheless, L2 rat lung epithelial cells are also used in this system because of their metabolising capacity. Tritiated proline is used as a label for the measurement of protein synthesis. In a comparison with radiolabelled leucine, proline was shown to be a more sensitive marker in HFL1 cells (Barnes et al., 1990). This was partly due to the fact that the culture medium contained leucine but not proline. Furthermore, proline is over-represented in collagenous proteins, and collagen synthesis was shown to be affected more specifically than other proteins in this cell strain. Leucine, on the other hand, is under-represented in protein manufactured by foetal lung fibroblasts. Its frequency in 53 analysed proteins was found to be approximately 7% (Doolittle, 1979).

CHEMICALS TESTED

On HFL 1: Acetaldehyde Cycloheximide Ethanol Ammonium chloride Prostaglandin E1 Leupeptin Hydroxynorvaline TLCK Puromycin Cis-hydroxyproline Paraquat On L2 : 50 chemicals of the MEIC study (Bondesson, I. et al., 1989)

TEST STATUS

The MEIC (multicenter evaluation of in vitro cytotoxicity) project aims to evaluate combinations of cytotoxicity tests, together with toxicokinetic and physicochemical data, for their relevance to human toxicity. The predictive capability of the cytotoxicity tests will be compared with that of rodent tests in vivo. An initial set of 50 chemicals to be tested was chosen on the basis of available human toxicity data, and laboratories were encouraged to test this set of chemicals in their in vitro cytotoxicity assays. Data from 38 cytotoxicity assays for the first ten MEIC compounds has been analysed, the `Lung Cell Assay' as described in this protocol was one of the 38 tests analysed ( Hellberg et al., 1990). One of the conclusions from this preliminary assessment was that analysis of the cytotoxicity test data collected for the first ten MEIC compounds indicate that the data are relevant for human toxicity. The MEIC report mentioned above describes the validation of the method (not the cell line) in collaboration with other laboratories. Chemicals 11-30 are currently being validated and the article is in press. Chemicals 31-50 have been tested and submitted to the MEIC committee but will not undergo validation until 1993. Both the HFL1 and L2 cell lines are in in-house use, and L2 is currently undergoing interlaboratory validation.

REFERENCES

  1. Barile F.A., Ripley-Rouzier C., Siddiqi Z. & Bienkowski R.S. (1988) Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts. Archs Biochem. Biophys. 265, 441-446.
  2. Barnes Y., Houser S. & Barile F.A. (1990) Temporal effects of ethanol on growth, thymidine uptake, protein and collagen production in human foetal lung fibroblasts. Toxic. in Vitro 4, 1-7
  3. Bondesson I., Ekwall B., Hellberg S., Romert L., Stenberg K. & Walum E. (1989) Meic-a new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests. Cell Biology and Toxicology, 5 No.3, 331-347.
  4. Breul S.D., Bradley K.H., Hance A.J., Schafer M.P., Berg R.A. & Crystal R.G. (1980) Control of collagen production by human diploid lung fibroblasts. J. Biol. Chem. 255, 5250-5260.
  5. Devereux T.R., Jones K.G., Bend J.R., Fouts J.R., Statham C.N. & Boyd M.R. (1982) In vitro metabolic activation of the pulmonary toxin 4-ipomeanol, in nonciliated bronchiolar epithelial (Clara) and alveolar type II cells isolated from rabbit lung. J. Pharmac. exp. Ther. 220, 223-227.
  6. Doolittle R.F. (1979) In: The proteins (eds. Neurath, H. and Hill, R.L.) Academic Press; New York, 1979 pp. 1-118. Hayflick L. (1965) The limited in vitro lifetime of human diploid cell strains. Expl. Cell Res. 37, 614-636.
  7. Hellberg, S., Eriksson, L., Jonsson, J., Lindgren, F., Sjostrom, M. & Wold, S. 1990 Analogy models for prediction of human toxicity. ATLA, 18, 103-116
  8. Jacobs J.P., Jones C.M. & Baillie J.P. (1976) Characteristics of a human diploid cell designated MRC-5. Nature (London) 227, 168-170.
  9. Barile F.A., Siddiqi Z., Ripley-Rouzier C. & Bienkowski R.S. (1989) Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts. Archs Biochem. Biophys. 270, 294-301.
  10. Barile F.A., Guzowski D.E., Ripley C., Siddiqi Z. & Bienkowski R.S. (1990) Ammonium chloride inhibits basal degradation of newly synthesised collagen in human fetal lung fibroblasts. Archs Biochem. Biophys. 276, 125-131.

IP-48 © February 1992