Summary.-An experimental model of pulmonary fibrosis has been developed by dosing rats with one-fifth the LD50 dose of the herbicide paraquat on 5 ...
Br. J. exp. Path. (1979) 60, 653
CHANGES IN TRANSGLUTAMINASE ACTIVITY IN AN EXPERIMENTAL MODEL OF PULMONARY FIBROSIS INDUCED BY PARAQUAT M. GRIFFIN, L. L. SMITH* AND J. WYNNE From the Department of Life Sciences, Trent Polytechnic, Nottingham, and the *Central Toxicology Laboratories, I.C.I. Limited, Alderley Park, Macclesfield, Cheshire Received for publication June 26, 1979
Summary.-An experimental model of pulmonary fibrosis has been developed by dosing rats with one-fifth the LD50 dose of the herbicide paraquat on 5 consecutive days. Approximately 50% of the rats died within 4 days of the completion of dosing, showing macroscopic changes and wet weight increases in the lung consistent with severe oedema. Those animals which died between Days 4 and 10 had markedly increased levels of hydroxyproline in the lung, maximum at Day 6, and increased prolyl hydroxylase activity, maximum at Day 4. These changes, together with an increase in thymidine incorporation into DNA, and increased lung DNA content, were consistent with the development of fibrosis. Measurement of transglutaminase activity in the lung showed marked increases at Days 4 and 10 after completion of dosing. This activity paralleled closely the changes in prolyl hydroxylase activity and became increasingly associated with particulate protein present in the "nuclear pellet" fraction. The presence of zymogen plasma transglutaminase trapped in lung homogenates could not be demonstrated but the contribution by the active plasma transglutaminase (Factor XIIIa) to increases shown at Day 4 cannot be ruled out. HUMAN PULMONARY FIBROSIS represents a reaction of the lungs to a wide variety of noxious stimulae (Witschi, 1977). In chronic cases tissue repair processes appear to escape normal regulatory control and lead to increased cellularity, deposition of fibrous tissue and eventual consolidation of the lung. One such compound causing this effect is the herbicide paraquat, 1,1'-dimethyl-
4,4'-bipyridilium dichloride (Bullivant, 1966; Clark, McElligott and Hurst, 1966). Pulmonary lesions resulting from paraquat poisoning are thought to progress through 2 phases, an acute oedematous inflammatory phase, followed by a more hypercellular fibrotic phase (Smith and Rose, 1977). With paraquat as the injurious agent, several animal models have been used to study experimentally induced pulmonary
fibrosis. Attempts to quantitate these models biochemically have resulted in a series of contradictory results when collagen metabolism was used as the prime marker. Significant changes in either prolyl hydroxylase activity or lung collagen content appear to differ according to the type of dosing regime employed (Hollinger, Zuckermann and Giri, 1978; Hollinger and Chvapil, 1977; Autor and Schmitt, 1977; Thompson and Patrick, 1978). In this report we have used biochemical studies to quantitate a fibrotic model developed using a paraquat dosing regime whereby a single LD50 dose was divided into 5 consecutive daily doses. In order to quantitate the fibrotic phase further and establish another potential marker of this phase, the importance of a group of enzymes, the transglutaminases, has been investigated in this model.
Correspondence: Dr M. Griffin, Department of Life Sciences, Trent Polytechnic, Nottingham NG1 4BU, England.
654
M. GRIFFIN, L. L. SMITH AND J. WYNNE
The transglutaminases are a group of amidotransferases which catalyse a calcium-dependent acyl-transfer reaction in which the y-carboxamide groups of peptide glutamine residues are the acyl donors. The acyl acceptors are various primary amines including the E-amino group of peptide lysine making cross-links between proteins possible (Mycek et al., 1959; Chung, 1972). The role of the plasma transglutaminase or Factor XIII in wound healing and haemostasis is well documented (Beck, Duckert and Ernst, 1961; Chen and Doolittle, 1971). The function of another group of transglutaminase enzymes widely distributed in mammalian tissues is poorly understood (Chung, 1972). The ability of the transglutaminase enzymes to cross-link fibrinogen and fibrin has led to investigations of their involvement in atherosclerosis (Laki, Benko and Farrel, 1972), hyaline membrane disease (Ambrus et al., 1968) and tumour metastatic growth (Yancey and Laki, 1972) Recent evidence demonstrating their ability to cross-link collagen (Soria, Soria and Boulard, 1975) and the cell surface glycoprotein fibronectin (Birckbichler and Patterson, 1978; Mosher, 1978) suggests they may also play a role in the organization of the connective tissue matrix. Relatively high levels of tissue transglutaminase have been found in both human and rat lung (Chung, 1972; Griffin et al., 1978). Subcellular localization of the rat lung enzyme has demonstrated that a large proportion of enzymic activity is associated with a fraction containing cell membranes and connective tissue debris (Griffin et al., 1978). These factors have prompted us to investigate further the importance of transglutaminase enzymes in a biochemically quantitated experimental model of pulmonary fibrosis. MATERIALS AND METHODS
Mlaterials. Paraquat dichloride was supplied by the Central Toxicology Laboratories, I.C.I. Ltd, Macclesfield. (1,4 14C)-putrescine, (3,4
3H)-proline and (6 3H)-thymidine were supplied by the Radiochemical Centre, Amersham. Animals. Female SPF rats (Sprague-Dawley strain) weighing 150-200 g were uised in all experiments. Food and water were given ad
libitum. Dosing of animals.-Paraquat dichloride, dissolved in sterile isotonic saline, was administered s.c. on 5 consecutive days using doses of 1/5 LD5o (5 mg paraquat ion/kg body wt). Controls were injected in the same way with isotonic saline. The LD50 dose of paraquat was determined by injecting rats s.c. with various doses of paraquat and scoring survival after 14 days. The mortality rate after dosing was determined by counting the number of dead animals every 24 h after the last injection of paraquat. Animals were killed by anaesthetizing with halothane and cutting the dorsal aorta. Lungs were removed, perfused with cold 0-15M NaCl, cut into small pieces and homogenized in 3 volumes of 0-25M sucrose/lmM EGTA/ImM Tris-chloride (pH 7.4) with a mechanically driven Potter-Elvejhem homogenizer. The homogenate was centrifuged at 600 x g (r av. 28 cm) for 10 min and the resulting supernatant decanted. The pellet was then rehomogenized in an equal volume of sucrose medium and combined with the first supernatant to produce the final homogenate. For some studies a sample of the homogenate was recentrifuged at 600 x g (r av. 28 cm) for 10 min to provide a "cytoplasmic fraction" and a "nuclear pellet" which was resuspended in an equal volume of sucrose medium for further analysis. For prolyl hydroxylase measurements lungs were homogenized with the addition of 50,M dithiothreitol and 50,g/ml phenylmethylsulphonyl fluoride. These homogenates were spun at 9000 x g (r av. 8 cm) for 15 min and the supernatant used for assay. All procedures were carried out at 4°. For hydroxyproline determinations homogenates were dialysed against distilled water before analysis to remove sucrose.
Enzyme assays. Transglutaminase activity was measured by a modification of the "filter
paper" assay of Lorand, Campbell-Wilkes and Cooperstein (1972). The reaction mixture incubated at 370 contained in a final volume of 0-1 cm3, 50-400 ,ug of protein, 5 mm CaCl2, 3.85 mM dithiothreitol, 500 /Ag of N,N'-dimethylcasein, 1-2 mm putrescine containing 2-5 ,Ci of (1,4 14C)-putrescine (sp. act. 62 mCi/mmole) and 30 mm Tris-chloride buffer (pH 7.4). Controls were run substituting 2-5 mM EDTA for CaC12. Samples of 10 M1 were taken from the reaction at appropriate intervals, enabling a linear rate of putrescine incorporation to be measured. Radioactive samples were counted in a Packard (Model 3330) Scintillation spectrometer using a scintillation mixture containing
PARAQUAT-INDUCED PULMONARY FIBROSIS
3 g of 2-5-diphenyloxazole (PPO) and 0 3 g of 1,4-di-(2-(5-phenyloxazoyl)-)-benzene (POPOP) per litre of toluene. To investigate the presence of zymogen plasma transglutaminase in the lung, homogenates were preincubated with 25 N.I.H. units of thrombin in 30mM Tris-chloride buffer (pH 7.4) containing 5mM CaCl2 for 20 min before assay. Prolyl hydroxylase activity was assayed by a modification of the method of Hutton and Udenfriend (1966). The method is based on the release of tritium into the incubation medium when a peptidyl (3,4 3H)-proline-rich, hydroxyprolinedeficient substrate prepared from 10-day chick embryo is incubated with the enzyme and cofactors aerobically. Each assay mixture contained in a final volume of 0 75 ml, 2 mM ascorbic acid, 1 mm ferrous ammonium sulphate, 2 mm cx-ketoglutarate, 0 5-1 mg of supernatant protein and 100 mm Tris-chloride buffer (pH 7.4). Control incubations were performed containing no ac-ketoglutarate. Reactions were started by addition of labelled substrate (12,000 ct/min) and incubated aerobically at 370 for 60 min. Reactions were terminated by addition of trichloroacetic acid to a final concentra-
tion of 5 % (w/v). The resulting tritiated water formed was collected by vacuum distillation at 60° and 0 5 ml of this made up to 1 ml with water and added to 10 ml of Instagel (Packard Instruments Limited). Radioactivity was measured by scintillation spectroscopy and activity expressed as ct/min. Protein was determined by the method of Lowry et al. (1951). Measurement of thymidine incorporation into DNA.-At various times after administration of paraquat or saline the rats were given 30 /LCi (6 3H)-thymidine/200 g body wt by i.p. injection. Animals were killed 1 h later, and their lungs removed and homogenized by the procedures outlined above. The DNA was precipitated by the addition of ice-cold trichloroacetic acid to give a final concentration of 5%
(w/v).
The precipitate was washed twice in ice-cold 5% (w/v) trichloroacetic acid and twice in ethanol 95% (w/v) at room temperature. The DNA was then hydrolysed in 0-5M perchloric acid for 20 min at 900. A portion of this (1 ml) was mixed with 10 ml of Instagel and the radioactivity measured in a Packard (Model
120
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70 14
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0 1 2 3 4 5 6 7 8 Days after dosing FIG. 1.-Percentage changes in body weights of rats during and after paraquat dosing, and the mean values for body weights of control animals (-). -
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655
M. GRIFFIN, L. L. SMITH AND J. WYNNE
656
sign of paraquat toxicity. This situation was found to exist in the experimental standard. model demonstrated in these results and The DNA determinations. present Chemical
3330) Scintillation counter. Counting efficiency was measured by the addition of an internal
in 1 ml of hydrolysate was measured by a modification of the diphenylamine method (Richards, 1974). The hydroxyproline content of lung was measured by the method of Prockop and Udenfriend (1960).
30
~20
T
RESULTS
Previous studies in which a single LD50 dose of paraquat for the development of a fibrotic model was used demonstrated quite clearly that a split population of animals develops after dosing (Smith and Rose, 1977). One population demonstrated the typical characteristics of paraquat poisoning while another population consisted of "typical survivors" showing little
110
2
1
Days c'ter
7
6
5
3
8
9
dos0rg
FIG. 2. Mortality of rats after paraquat dosing, determined by counting the number of dead animals every 24 h after the last injection of paraquat.
TABLE I.-Lung Wet Weights in Paraquat-treated Animals Percentage of
Values as percentage of control mean
Sampling time (days after completion of dosing)
Mean
Highest
2 4 6 8 10
222 227 201 135 184
346 295 253 186 279
animals in group with values greater than
Lowest 160 118 98 110 110
hiighest control value 100 91 77 60 83
Control mean for lung wet weight = 1-185 + 0-20 g. Number of animals per group varied from 5-18.
TABLE II.-Levels of Collagen and its Synthesis in Lungs of Paraquat-treated Animals Sampling time (days after completion of dosing)
Prolyl hydroxylase activity (units/lung)
Lung hydroxyproline (nmol/lung)
2 4 6 8 10 2 4 6 10
Values as a percentaige of control mean Mean 64 153 116 96 137 108 79 251 153
Highest 86 158 184 108 157 157 112 295 182
Lowest 44 149 61 72 121 76 28 207 138
Percentage of animals in group with v,alues greater than highest control value 0 100 33 0 100 33
25
100 75
Unit prolyl hydroxylase activity = ct/min tritium released/h. Control mean value for prolyl hydroxylase activity (units/lung) = 1-67 x 106 + 0-226 x 106. Control mean for lung hydroxyproline content (nmol/lung) = 5-67 + 1-095. Number of animals per group varied from 3 to 6.
657
PARAQUAT-INDUCED PULMONARY FIBROSIS
may be demonstrated by rat body weights during and after the period of paraquat dosing (Fig. 1). For this reason results are generally expressed as mean values, the highest and lowest values, and the percentage of animals above the highest control value. Statistics were not performed on these results for reasons stated above.
Effect of paraquat on rat lung Approximately 50%o of the animals died between Days 2 and 4 after paraquat dosing had been discontinued (Fig. 2). Lung wet weights were shown to have increased by about 120%o over those of controls between these days (Table I). Quantitation of lung collagen metabolism In order that lung collagen metabolism might be assessed, prolyl hydroxylase activity was used as a measure of collagen synthesis (Autor and Schmitt, 1977) and hydroxyproline levels as a measure of lung collagen content. Lung hydroxyproline levels indicated a l50%O increase over controls at Day 6 and a 5000 increase at Day 10 (Table II). This correlated well with increases in prolyl hydroxylase activity (units/lung) at Days 4 and 10 (50 and 37%o increases over control values respectively; Table II). Units of activity expressed per mg of protein
showed no increases over control values (Table III).
Quantitation of the hypercellular phase The hypercellular response occurring during the fibrotic phase was assessed by measurement of DNA synthesis using the incorporation of (6 3H)-thymidine into DNA. Using this regime it was found that DNA synthesis was at its highest on Day 4 and fell gradually till Day 10 (Table IV). The magnitude of this increase was relatively low compared to that seen by Smith and Rose (1977) but in agreement with the results of van Osten and Gibson (1974). In each of these cases a single dosing regime was used. The total DNA level, which increased to its maximum at Day 6 (150% over control values), correlated well with the maximum increase in DNA synthesis at Day 4 (Table IV). Effect of paraquat upon lung transglutaminase activity Transglutaminase activity expressed in terms of whole-lung activity was increased over that of control animals by 100-150% at Days 4 and 10 after dosing (Table V); 66-83% of the animals showed higher values than the highest control.
TABLE III. Specific Activity of Prolyl Hydroxylase in Lungs of Paraquat-treated Animals Sampling time (days after completion of dosing) Prolyl hydroxylase activity per mg protein Prolyl hydlroxylase activity per mg DNA
9
4 6 10 4 6 10
Values as a percentage of control mean
M\ean 59
Highest 79
81 98
103 129 112
Lowest 41 71 42 86
43 91 46 80
56 94 74 92
29 65 24 71
99
Unit of prolyl hydroxylase activity= ct/min tritium released/h. Control mean for units/mg protein = 12100 + 1630. Control mean for units/mg DNA = 3-34 x 105 + 0l IO X 105. Number of animals per group was 3.
Percentage of animals in group witlh values greater than highest control value 0 0 33 0 0 0 0 0
658
M. GRIFFIN, L. L. SMITH AND J. WYNNE
TABLE IV.-Levels of DNA and its Synthesis in Lungs of Paraquat-treated Animals Sampling time (days after completion of dosing)
[3H]-Thymidine incorporation into lung DNA Lung DNA content
Values as a percentage of control mean
4 6 10
141 182 149 137
Highest 190 187 208 147
Lowest 107 178 90 126
2 4 6 10
151 168 250 171
222 222 314 255
85 134 160 104
9
N- lean
Percentage of animals in group with values greater than highest control value 100 100 86
:33 67 50 100 67
Control mean for [3H]-thymidine incorporation into DNA (dpm/mg DNA) = 8344 + 1256. Control mean lung DNA content (mg DNA/lung) = 5-32 + 1-17. Number of animals per group varied from 6 to 15.
Transglutaminase activity expressed as units per mg of protein still showed an increase over control values at Days 4 and 10, although not as high as the total activity per lung might predict (Table V). Only 170% of the animals at Day 4 and 50%o at Day 10 showed values higher than the highest control. This dilution in specific activity (u/mg protein) was also demonstrated with prolyl hydroxylase at the same time period and is not unusual if one considers that a highly inflammatory phase involving an oedematous influx of
proteins is occurring between Days 2 and 4. Further dilutions occurring at Days 6 and 10 might be reflected by the increases in lung collagen content at these periods. The difficulties which are associated with measuring enzymic activities in injured lung tissue have caused other authors to express their results in terms of whole-lung activity only (Thompson and Patrick, 1978). No increase in transglutaminase activity was observed which might be contributed by plasma transglutaminase when lung
TABLE V. Transglutaminase Levels in Lungs of Paraquat-treated Animals Sampling time (days after completion of dosing) 2 Transglutaminase activity 4 (units/lung) Transglutaminase activity (units/mg DNA) Transglutaminase activity (units/mg protein)
Values as a percentage of control mean
6 10
Mean 120 200 143 246
Highest 197 464 237 338
Lowest 90 88 103 101
4 6 10
86 108 63 151
191 209 134 318
41 54 38 40
2 4 6
110 126 98
10
186
182 264 150 363
71 53 98 53
Percentage of animals in group with values greater than highest control value 47 67 90 83
27 17 0 5( 47 17 33 50
Control means for units/lung = 8006 + 960: units/mg DNA = 1557 + 440: units/mg protein Unit of enzyme activity= nmol of putrescine incorporated/h. Number of animals per group varied from 6 to 15.
=
57-0 + 10-5.
PARAQUAT-INDUCED PULMONARY FIBROSIS
659
TABLE VI.-Subcellular Distribution of Transylutaminarse Activity in Lungs of Paraquattreated Animals Sampling time (days after completion of dosing) 2 4 6 10
Percentage of animals in group Values as a percentage of with values control mean greater than highest control Mean Highest Lowest value 284 428 122 83 300 474 177 100 307 514 122 80 469 640 296 100 "Nuclear fraction" fraction"
Control mean for amount of enzymic activity in "Cytoplas Number of animals per group varied from 3 to 6.
=
1-083 + 0-215.
homogenates were activated by thrombin fibrosis in which the importance of a group before the reaction assay. of enzymes, the transglutaminases, could be assessed. The model chosen for this study was Differential distribution of transglutaminase activity developed using paraquat administered to The large amount of rat lung trans- rats in a split-dosing regime where the glutaminase activity associated with a LD50 was divided into 5 consecutive daily subeellular fraction containing cell mem- doses. The large mortality rate which branes and connective tissue matrix resulted from this model in the 2-4 days (Griffin et al., 1978) caused us to investigate following dosing may be likened to that in any differential change in this distribution other models in which a single LD50 dose after pulmonary injury by paraquat. was used (Smith and Rose, 1977). ApproxiNormal distribution ratios of trans- mately 50 % of the animals died at this glutaminase activity present in the 600g stage and considerable changes in the wet cell "Nuclear Pellet fraction" to that in weight of rat lungs were recorded. Death the rest of the cell "cytoplasmic fraction" of animals at this stage was probably due are of the order of 1 1:1. After paraquat to severe inflammatory changes in the lung damage an increase in activity present in (Smith and Rose, 1977; Thompson and the 600g pellet occurred between Days Patrick, 1978). The division of the LD50 2 and 10, the final distribution pattern dose into a split-dosing regime did not reaching 5-0:1 at Day 10 (Table VI). The appear to reduce this inflammatory phase, main differences demonstrated (results not as was originally intended. Biochemical markers used to measure shown) between the normal lung 600g pellet and the paraquat-treated lung 600g the fibrotic phase indicated that maximum pellet when viewed by electron microscopy collagen synthesis was occurring at Days 4 was that the injured lung had a general and 10 after dosing. This, in conjunction tendency to be less fully homogenized with maximum DNA synthesis occurring between Days 4 and 8. This is probably at Day 4, would suggest that many of the accounted for by the increased connective cells proliferating at this period were of the fibroblastic type. This result would tissue matrix at this period. agree with those of the combined biochemical and morphological studies of DISCUSSION Greenberg, Reiser and Last (1978), in The purpose of this investigation was to which a single dosing regime with paraquat produce a reliable model of pulmonary was used.
660
M. GRIFFIN, L. L. SMITH AND J. WYNNE
Similarities of prolyl hydroxylase activity (u/mg DNA) to that of control values at Day 4 would support the evidence that the cell population was probably heterogeneous. If the cell population was predominantly fibroblastic, then an increase in cells producing collagen rather than an increase in its rate of synthesis would account for the raised levels of prolyl hydrolase at this period (Hance et al., 1976). In contrast to the model demonstrated by Thompson and Patrick (1978), in which a single dosing regime was used, increased DNA content in paraquat-treated lung was observed, the increase being maximal at Day 6. The correlation between increased DNA synthesis at Day 4 and increased lung collagen content at Day 6 would suggest that death of animals at this stage was due to fibrosis, a result supported by macroscopic observation of the lungs. Increases of prolyl hydroxylase activity and hydroxyproline content over control values at Day 10 are not unusual considering that the lungs of these animals are likely to be undergoing repair, a process requiring a gradual increase in connective tissue matrix (Bradley, McConnell and Crystal, 1974). These animals are likely to survive the 'paraquat-induced trauma, an observation demonstrated by a gradual increase in their body weight at this stage (Fig. 1). The known catalytic properties of the transglutaminase enzymes, together with the large increases in activity demonstrated in this model, suggests their importance. Of particular interest is the parallel rise in enzymic activity with that of prolyl hydroxylase, an increase which either immediately precedes or coincides with maximum collagen deposition at Days 6 and 10 respectively. Their possible role in the organization of the connective tissue matrix cannot be ignored when one considers their ability to catalyse into polymeric aggregates proteins such as collagen, fibrin and fibronectin (Soria et al., 1975; Nyman and Duckert, 1975; Birckbichler
and Patterson, 1978). Furthermore their ability to incorporate primary amines (e.g. putrescine, histamine and spermine) into tissue proteins could result in proteins of altered physical states following pulmonary injury. The rise of transglutaminase in parallel with that of prolyl hydroxylase would tend to associate this acdtivity with a cell type capable of synthesizing collagen. This would include the mesenchymal fibroblasts, endothelial cells and the Type I pneumocytes (Hance et al., 1976). Recent evidence by Stenman and Vaheri (1978) has demonstrated that accumulations of fibroblasts at inflammatory sites show increased amounts of fibronectin. The close association of transglutaminase with fibronectin has been reported by Birckbichler and Patterson (1978) in WI-38 cells from human embryonic lung. It is recognized that associations between fibronectin, collagen and fibrin occur at the surface of the fibroblast (Bornstein and Ash, 1977), but the exact nature of these associations is still unknown. Interactions mediated via transglutaminase are a reasonable possibility and would correlate well with the increased amount of transglutaminase activity which becomes associated with the 600g pellet of homogenized paraquat-damaged lung. The inability of thrombin to show any increase in transglutaminase activity does not rule out the possibility that activated plasma transglutaminase (Factor XIIIa) may already be present and contribute to the increase over control values at Day 4. Influx of plasma proteins during the oedematous phase at Days 2-4 would increase the amount of this enzyme present at this stage. The results presented in this paper suggest an important role for the transglutaminase enzyme in the development of fibrosis. Whether the catalytic activity of the enzyme is contributing to a fibrotic matrix which cannot easily be degraded and hence aiding the onset of obliterative fibrosis is a question we are at present trying to answer.
PARAQUAT-INDUCED PULMONARY FIBROSIS
We would like to thank I.C.I. and the Science Research Council for financing this work. REFERENCES AMBRUS, C. M., WEINTRAUB, D., NISWANDER, K. & AMBRUS, J. L. (1968) Studies on the Ontogeny and Significance in Neonatal Disease of the Fibrinolysin System and of Fibrin Stabilizing Factor. Thromb. Diath. Haemorrh., 19, 599. AUTOR, A. P. & SCHMITT, S. L. (1977) Pulmonary Fibrosis and Paraquat Toxicity: In Biochemical Mechanisms of Paraquat Toxicity. Ed. A. P. Autor. London and New York: Academic Press. BECK, E., DUCKERT, F. & ERNST, M. (1961) The Influence of Fibrin-stabilizing Factor on the Growth of Fibroblasts in Vitro and Wound Healing. Throm. Diath. Haemorrh., 6, 485. BIRCKBICHLER, P. J. & PATTERSON, M. K. (1978) Cellular Transglutaminase, Growth and Transformation. Ann. N. Y. Acad. Sci., 312, 354. BORNSTEIN, P. & ASH, J. F. (1977) Cell Surfaceassociated Structural Proteins in Connective Tissue Cells. Proc. Natl. Acad. Sci., U.S.A., 74, 2480. BRADLEY, K. H., MCCONNELL, S. D. & CRYSTAL, R. G. (1974) Lung Collagen Composition and Synthesis. J. Biol. Chem., 249, 2674. BULLIVANT, C. M. (1966) Accidental Poisoning by Paraquat: Report of Two Cases in Man. Br. med. J., i, 1272. CHEN, R. & DOOLITTLE, R. F. (1971) y-y Crosslinking Sites in Human and Bovine Fibrin. Biochemistry, 10, 4486. CHUNG, S. I. (1972) Comparative Studies on a Tissue Transglutaminase and Factor XIII. Ann. N.Y. Acad. Sci., 202, 240. CLARK, D. G., McELLIGOTT, T. F. & HURST, E. W. (1966) The Toxicity of Paraquat. Br. J. industr. Med., 23, 126. GREENBERG, D. B., REISER, K. M. & LAST, J. A. (1978) Correlation of Biochemical and Morphological Manifestations of Acute Pulmonary Fibrosis in Rats Administered Paraquat. Chest, 74, 421. GRIFFIN, M., BARNES, R. N., WYNNE, J. & WILLIAMS, C. R. (1978) The Effects of Bleomycin and Copper Bleomycin upon Transglutaminase Enzymes. Biochem. Pharmacol., 27, 1211. HANCE, A. J., HORWITZ, A. L., COWAN, M. J., ELSON, N. A., COLLINS, J. F., BIENKOWSKI, R. S., BRADLEY, K. H., MCCONNEL-BREUL, S., WAGNER, W. M. & CRYSTAL, R. G. (1976) Biochemical Approaches to the Investigation of Fibrotic Lung Disease. Chest, 69, 257. HOLLINGER, M. A., ZUCKERMANN, J. E. & GIRI, S. N. (1978) Effect of Acute and Chronic Paraquat on
44
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Rat Lung Collagen Content. Res. Commun. Chem. Path. Pharm., 21, 295. HOLLINGER, M. A. & CHVAPIL, M. (1977) Effect of Paraquat on Lung Prolyl Hydroxylase Activity. Res. Commun. Chem. Path. Pharm., 16, 159. HUTTON, J. J. & UDENFRIEND, S. (1966) Soluble Collagen Prolyl Hydroxylase and its Substrates in Several Animal Tissues. Proc. Nat. Acad. Sci., U.S.A., 56, 198. LAKI, K., BENKO, S. & FARRELL, F. (1972) Clot Stabilization and Atherosclerosis. Ann. N. Y Acad. Sci., 202, 235. LORAND, L., CAMPBELL-WILKES, L. K. & COOPERSTEIN, L. (1972) A Filter Paper Assay for Transamidating Enzymes using Radioactive Substrates. Anal. Biochem., 50, 623. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951) Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193, 265. MOSHER, F. (1978) Crosslinking of Plasma and Cellular Fibronectin by Plasma Transglutaminase. Ann. N. Y. Acad. Sci., 312, 38. MYCEK, M. J., CLARK, D. D., NEIDLE, A. & WAELSCH, H. (1959) Amine Incorporation into Insulin as Catalysed by Transglutaminase. Arch. Biochem. Biophys., 84, 528. NYMAN, D. & DUCKERT, F. (1975) Factor XIII, Fibrin and Collagen. Thromb. Diath. Haemorrh., 34, 551. PROCKOP, D. J. & UDENFRIEND, S. (1960) A Specific Method for the Analysis of Hydroxyproline in Tissues and Urine. Anal. Biochem., 1, 228. RICHARDS, G. M. (1974) Modifications of the Diphenylamine Reaction giving Increased Sensitivity and Simplicity in the Estimation of DNA. Anal. Biochem., 57, 369. SMITH, L. L. & ROSE, M. S. (1977) A Comparison of the Effect of Paraquat and Diquat on the Water Content of Rat Lung and the Incorporation of Thymidine into Lung DNA. Toxicology, 8, 223. SORIA, A., SORIA, C. & BOULARD, C. (1975) Fibrin Stabilizing Factor (F XIII) and Collagen Polymerisation. Experientia, 31, 1355. STENMAN, S. & VAHERI, A. (1978) Distribution of a Major Connective Tissue Protein, Fibronectin, in Normal Human Tissues. J. exp. Med., 147, 1355. THOMPSON, W. D. & PATRICK, R. S. (1978) Collagen Prolyl Hydroxylase Levels in Experimental Paraquat Poisoning. Br. J. exp. Path., 59, 288. VAN OSTEN, G. K. & GIBSON, J. E. (1974) The Effect of Paraquat on the Biosynthesis of Deoxyribonucleic Acid, Ribonucleic Acid and Protein in the Rat. Fd. Cosmet. Toxicol., 13, 47. WITSCHI, H. (1977) Primary Pulmonary Responses to Toxic Agents. Crit. Rev. Toxicol., 5, 23. YANCEY, S. T. & LAKI, K. (1972) Transglutaminase and Tumour Growth. Ann. N.Y. Acad. Sci., 202, 344.
