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Mitodepressive, clastogenic and biochemical effects of (+)-usnic acid in mice
Journal of Ethnopharmacology,
33 ( I99
1) 2 I l-220
217
Elsevier Scientific Publishers Ireland Ltd.
Mitodepressive,
clastogenic and biochemical effects of (+)-usnic acid in mice
A.M. Al-Bekairi”, S. Qureshia, M.A. Chaudhrya’*, D.R. Krishnaa’** and A.H. Shahb “Quality
Control and Research Laboratory,
University,
Riyadh-11451
E.yperimental
and hCentral Laboratory
Animal
Care Centre. College of Pharmacy,
for Drug and Food Analysis,
P. 0. Bos 59082,
P.O.
Box 2457. King Saud
Riyadh-II525
(Saudi Arabia)
(Accepted December 19. 1990) Mice were treated orally with aqueous suspensions of (+)-usnic acid in a single dose of either 100 or 200 mg/kg. The effects on femur cells and proteins and on nucleic acids of liver cells were studied 24-72 h after treatment. (+)-Usnic acid was found to affect the proliferation of polychromatic erythrocytes possibly by interference with RNA biosyntheis. The slight increase in the micronucleated polychromatic erythrocytes without affecting DNA synthesis suggestsan effect of usnic acid on spindle apparatus. Key words: (+)-usnic acid; lichen; mitodepression: clastogenic effects.
Introduction
Materials and Methods
Lichen species are used in many countries medicinally (Trease and Evans, 1978; Kirtikar and Basu, 1984; Huovinen, 1988). Usnic acid, the most abundant lichen constituent (Shibamoto and Wei, 1984), is known to possess antibacterial and antifungal activity (Kupchan and Kopperman, 1975; Windholz and Budavari, 1984). (-)-Usnic acid, previously reported to have some activity in the Lewis lung tumor and in the murine P388 leukemia test systems (Takai et al., 1979), recently was found to be cytotoxic in the Allium test (Huovinen and Lampero, 1988). (+)-Usnic acid has been shown to be immunologically active in allergic contact dermatitis and to have profound antitubercular acitivity (Shibata, 1978). The present study was undertaken to evaluate the mitodepressive and clastogenic potential of (+)usnic acid as a part of safety evaluation before its clinical trial.
Animal stock Male Swiss albino mice (SWR, home bred) aged 5-6 weeks, weighing 25-30 g, fed on Purina Chow diet and water ad libitum were used in this study. An arbitrary dose of 200 mg/kg was found to deplete polychromatic erythrocytes in a pilot study; therefore. in the present study (+)-usnic acid (Sigma Chemical Co., St. Louis, MO) was given orally suspended in water to test animals in single doses of either 100 or 200 mg/kg. Animals in the control group received an equivalent volume of distilled water.
Correspondence to: S. Qureshi, Quality Control and Research
Laboratory,
Experimental Animal
Care Centre, College of
P.O. Box 2457, King Saud University, Pharmacy, Riyadh- I I45 I, Saudi Arabia. Present address: *Department of Zoology, Govt. College. Lahore, Pakistan. **College of Pharmaceutical Sciences, Kakatiya University. Warangal, India.
0378-8741603.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Micronucleus test The micronucleus test is reliable in vivo test for detecting potential clastogens (Hayashi et al., 1984) and is highly suitable for routine toxicological screening (Schmid, 1975). A total of 45 mice were randomly allotted to the different control and treatment groups. The test procedure described by Schmid (1975) was followed. Animals in the test group were administered 100 or 200 mg/kg of test drug orally and killed 24, 48 and 72 h after the treatments. The adhering soft tissues and epiphyses of both tibiae of each animal were removed. Marrow was aspirated from the femur into foetal calf serum and transferred to a centri-
218
TABLE 1 EFFECT OF (+)-USNIC ACID ON THE RATIO OF POLYCHROMATIC Oral treatment (dose)
AND NORMOCHROMATK
CELLS IN MICE
Ratio of polychromatic and norrnochromatic cells
Distilled water (IO ml/kg) Usnic acid t 100mg/kg) Usnic acid (200 mg/kg)
+24 h
+48 h
I.15 f 0.08
0.94
0.86
0.89 zt 0.10
1.05 f 0.02
0.97 * 0.08
0.99 * 0.07
l
0.12
0.74 f 0.03*
+72 h 0.06
l
I.15
f
0.10
Tabular values represent the mean f S.E.M. of 5 mice/group. Significant from the distilled water control: *P < 0.001.
fuge tube. After centrifugation at 1000 rev./min for 5 min, the supernatant was discarded, the residual cells spread on slides, air dried and stained in May Gruenwald solution followed by Giemsa. The coded slides were screened for the presence of micronuclei in polychromatic erythrocytes, which indicates non-disjunction, chromosomal breaks and structural or numerical changes in the chromosomes. The mitodepression was calculated as a ratio of polychromatic/normochromatic cells.
were determined according to the methods described earlier (Wannemacher et al., 1965; Munro and Fleck, 1969). Purified calf liver RNA was used as standard and samples extracted for DNA were compared with purified calf thymus DNA. As standards, bovine serum albumin, RNA and DNA obtained from Sigma (St. Louis) were used. The different parameters studied were subjected to statistical analysis by Student’s t-test. Results and Discussion
Biochemical
procedures
Livers from the same animals were quickly excised, frozen in liquid nitrogen and stored at -20°C till analyzed for total proteins and nucleic acids (RNA and DNA). Total proteins were estimated by the modified Lowry method of Schacterle and Pollack (1973). Bovine serum albumin was used as a standard. Nucleic acids
The administration of (+)-usnic acid was found to decrease the proliferation of polychromatic erythrocytes at 24 and 48 h of exposure at both doses (100 and 200 mg/kg). However, the depletion was statistically significant (P c 0.001) at the highest dose only after an exposure of 24 h (Table 1).
TABLE 2 EFFECT OF (+)-USNIC ACID ON PROTEIN AND NUCLEIC ACID CONTENTS OF LIVER IN MICE Oral treatment (dose) Distilled water (IO ml/kg) Usnic acid t loo mdkg) Usnic acid t200 mg/kg)
Protein (mg/lOO mg)
RNA WI00
DNA (lrg/lOfJ mg)
mg)
+24 h
+48 h
+72 h
+24
h
+48 h
+72 h
+24 h
+48
h
+72 h
12.2 l 2.2 12.1 l 1.3 12.4 f I.9
II.8 zt I.8 9.4 f 0.5 9.4 l I.3
II.9 l 1.2 12.3 l I.0 I I.6 * I.8
599 l 59 503 f 48 487 f 70
616 f 52 569 f 63 599 * 73
615 zt 65 625 * 73 609 zt 62
308 l 30 321 f 32 315 l 6t3
312 f 45 325 *40 311 * 49
315 l 34 310 f 42 319 f 38
Tabular values represent the mean f S.E.M. of 5
mice/group.
219
TABLE EFFECT
3 OF (+)-USNIC
Oral treatment
ACID
ON THE
FREQUENCY
+24 h
OF MICEONUCLEI
IN FEMORAL
CELLS
OF MICE
+72 h
+48 h
(dose) Polychromatic cells screened
Micronucleated
Polychromatic
Micronucleated
Polychromatic
Micronucleated
polychromatic
cells screened
polychromatic
cells screened
polychromatic
(N)
(‘%I)
(N)
(‘%I)
(N)
(‘X)
Distilled water (IO ml/kg)
6560
0.28
f
0.05
5051
0.22
f
0.09
4518
0.22
f
0.02
Usnic acid
4500
0.37
& 0.10
4667
0.57
*
0.15
4943
0.33
*
0.05
4606
0.48
+ 0.09
4001
0.41
f
0.07
4448
0.39
l
0.12
(100 mg/kg) Usnic acid (200 mgkg) Percentile values represent the mean
f
S.E.M.
of 5 mice/group.
The results on the quantification of proteins nucleic acid levels of liver cells in the same animals showed an apparent reduction in the levels of RNA at 24 and 48 h. Proteins appeared to be reduced at 48 h only for both doses (Table 2). Although the depletions were all statistically insignificant, the effect was consistent enough for the authors to consider that (+)-usnic acid may block the translation process by interferring with RNA biosynthesis (Tata, 1974; Brawerman, 1974; Lone et al., 1986; Al-Ahdal et al., 1988). The observed mitodepressant effect on the polychromatic erythrocytes may be due to an effect on mRNA molecules. Although comparable studies on (+)-usnic acid are scanty in literature, our results are in agreement with the findings on (-)usnic acid which was shown to be cytotoxic in some in vitro tests (Takai et al., 1979; Huovinen and Lampero, 1988). The effect of (+)-usnic acid on clastogenic activity was also studied using the femur cells of mice. There was an apparent increase in the micronucleated polychromatic erythrocytes at 24 and 48 h for both doses (Table 3). However, the increases were statistically insignificant and values returned to normal at 72 h. Simultaneous observations on DNA levels in liver cells showed no significant changes between the control and treated groups (Table 2). The increase in micronucleated polychromatic erythrocytes without affecting the level of DNA suggests a toxic effect of (+)-usnic
acid on the spindle apparatus (Schmid, 1975). Further work on the anti-cancer!cytotoxic potential of (+)-usnic acid and the clastogenicity of the two isomers is required to elucidate the exact mode of action of these compounds. Acknowledgement
We are thankful to Dr. A.V.N.A. Rao for his kind remarks and suggestions, References Al-Ahdal.
M.N..
Cytotoxicity
McGarry.
T.J.
and
Hannan.
M.A.
(1988)
of khat (Cutha edulis) extract on cultured mam-
malian cells: Effects on macromolecule
biosynthesis.
Mu/u-
tion Reseurch 204. 621-642. Brawerman.
G.
(1974)
Eukaryotic
Review cf Biochemistry Hayashi.
massenger
RNA.
Annual
43. 62 1-642.
M.. Sofuni. T. and Ishidate. M.. Jr. (1984) A pilot ex-
periment
for the micronucleus
multidose levels method. Huovinen,
test. The multi-sampling
Mutution
K. (1988) The content of protocetraric
ferent decoctions of Cetruriu islundicu. 36th on Mrdicinul
at
Reseurch 141, 165-169.
Plunt Reseurch (Freiburg,
acid in dif-
Annwl
12-16
Congress
September),
p. 31, (P2--12). Huovinen,
K. and Lampero.
inhibitor
M. (1988) Usnic acid as a mitotic
in the Album test. 361h Annuul Congress on Medici-
nal Plum
Research
(Freiburg.
12-16
September),
p. 31
and Basu. B.D. (1984) lncliun Medicinul
Plunrs.
(P2--12). Kirtikar.
K.R.
Lalit Mohan
Basu Pub.. Allahabad.
India. pp. 276&2761.
Kupchan. S.M. and Kopperman. H.L. (1975) Usnic acid: Tumor inhibitor isolated from lichen. Esperientiu 31. 625-626.
220 Lone, K.P., Chaudry, M.A. and Matty, A.J. (1986) Effect of thyroid hormone and actinomycin-D on protein and nucleic acids metabolism in carp liver and muscle. Pakistan Journal of Zoology 18, 297-309.
Munro, H.N. and Fleck, A. (1969) Analysis of tissue and body for nitrogenous constituents. In: H.N. Munro (Ed.), Mammalian Protein Metabolism. Vol. 3, Academic Press, London, pp. 423-525. Schacterle, G.R. and Pollack, R.L. (1973) A simplified method for quantitative assay for small amounts of proteius in biological materials. Analyrical Biochemistry 51, 654-655. Schmid, W. (1975) The micronucleus test. Mutation Research 31, 9-15.
Shibamoto, T. and Wei, C.I. (1984) Mutagenicity of lichen constituents. Environmental Mutagenesis 6, 757-762. Shibata, S. (1978) Lichen Substances. In: F. Korte and M.
Goto (Eds.), Natural Compounds. Georg Thieme Pub., Stuttgart, pp. 208-223. Takai, M.. Uehara, Y. and Beisler, J.A. (1979) Usnic acid derivatives as potential antineoplastic agents. Journal of Medicinal Chemistry 22. 1380-l 384. Tata, J.R. (1974) Growth and developmental action of thyroid hormones by actinocycin-D and puromycin. Nature 197. 1167-l 168. Trease, G.E. and Evans, WC. (1978) Pharmacognosy. Bailliere Tindall, London, pp. 621-623. Wannemacher, R.W., Jr., Bank, W.L.. Jr. and Wunner. W.H. (1965) Use of single tissue extract to determine cellular protein and nucleic acids concentration and rate of amino acid incorporation. Analytical Biochemistry I I, 320-326. Windholz, M. and Budavair, S. (1983) The Merck Index. 10th Edn. Merck and Co. Inc., Rahway NJ, p. 1414.
33 ( I99
1) 2 I l-220
217
Elsevier Scientific Publishers Ireland Ltd.
Mitodepressive,
clastogenic and biochemical effects of (+)-usnic acid in mice
A.M. Al-Bekairi”, S. Qureshia, M.A. Chaudhrya’*, D.R. Krishnaa’** and A.H. Shahb “Quality
Control and Research Laboratory,
University,
Riyadh-11451
E.yperimental
and hCentral Laboratory
Animal
Care Centre. College of Pharmacy,
for Drug and Food Analysis,
P. 0. Bos 59082,
P.O.
Box 2457. King Saud
Riyadh-II525
(Saudi Arabia)
(Accepted December 19. 1990) Mice were treated orally with aqueous suspensions of (+)-usnic acid in a single dose of either 100 or 200 mg/kg. The effects on femur cells and proteins and on nucleic acids of liver cells were studied 24-72 h after treatment. (+)-Usnic acid was found to affect the proliferation of polychromatic erythrocytes possibly by interference with RNA biosyntheis. The slight increase in the micronucleated polychromatic erythrocytes without affecting DNA synthesis suggestsan effect of usnic acid on spindle apparatus. Key words: (+)-usnic acid; lichen; mitodepression: clastogenic effects.
Introduction
Materials and Methods
Lichen species are used in many countries medicinally (Trease and Evans, 1978; Kirtikar and Basu, 1984; Huovinen, 1988). Usnic acid, the most abundant lichen constituent (Shibamoto and Wei, 1984), is known to possess antibacterial and antifungal activity (Kupchan and Kopperman, 1975; Windholz and Budavari, 1984). (-)-Usnic acid, previously reported to have some activity in the Lewis lung tumor and in the murine P388 leukemia test systems (Takai et al., 1979), recently was found to be cytotoxic in the Allium test (Huovinen and Lampero, 1988). (+)-Usnic acid has been shown to be immunologically active in allergic contact dermatitis and to have profound antitubercular acitivity (Shibata, 1978). The present study was undertaken to evaluate the mitodepressive and clastogenic potential of (+)usnic acid as a part of safety evaluation before its clinical trial.
Animal stock Male Swiss albino mice (SWR, home bred) aged 5-6 weeks, weighing 25-30 g, fed on Purina Chow diet and water ad libitum were used in this study. An arbitrary dose of 200 mg/kg was found to deplete polychromatic erythrocytes in a pilot study; therefore. in the present study (+)-usnic acid (Sigma Chemical Co., St. Louis, MO) was given orally suspended in water to test animals in single doses of either 100 or 200 mg/kg. Animals in the control group received an equivalent volume of distilled water.
Correspondence to: S. Qureshi, Quality Control and Research
Laboratory,
Experimental Animal
Care Centre, College of
P.O. Box 2457, King Saud University, Pharmacy, Riyadh- I I45 I, Saudi Arabia. Present address: *Department of Zoology, Govt. College. Lahore, Pakistan. **College of Pharmaceutical Sciences, Kakatiya University. Warangal, India.
0378-8741603.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Micronucleus test The micronucleus test is reliable in vivo test for detecting potential clastogens (Hayashi et al., 1984) and is highly suitable for routine toxicological screening (Schmid, 1975). A total of 45 mice were randomly allotted to the different control and treatment groups. The test procedure described by Schmid (1975) was followed. Animals in the test group were administered 100 or 200 mg/kg of test drug orally and killed 24, 48 and 72 h after the treatments. The adhering soft tissues and epiphyses of both tibiae of each animal were removed. Marrow was aspirated from the femur into foetal calf serum and transferred to a centri-
218
TABLE 1 EFFECT OF (+)-USNIC ACID ON THE RATIO OF POLYCHROMATIC Oral treatment (dose)
AND NORMOCHROMATK
CELLS IN MICE
Ratio of polychromatic and norrnochromatic cells
Distilled water (IO ml/kg) Usnic acid t 100mg/kg) Usnic acid (200 mg/kg)
+24 h
+48 h
I.15 f 0.08
0.94
0.86
0.89 zt 0.10
1.05 f 0.02
0.97 * 0.08
0.99 * 0.07
l
0.12
0.74 f 0.03*
+72 h 0.06
l
I.15
f
0.10
Tabular values represent the mean f S.E.M. of 5 mice/group. Significant from the distilled water control: *P < 0.001.
fuge tube. After centrifugation at 1000 rev./min for 5 min, the supernatant was discarded, the residual cells spread on slides, air dried and stained in May Gruenwald solution followed by Giemsa. The coded slides were screened for the presence of micronuclei in polychromatic erythrocytes, which indicates non-disjunction, chromosomal breaks and structural or numerical changes in the chromosomes. The mitodepression was calculated as a ratio of polychromatic/normochromatic cells.
were determined according to the methods described earlier (Wannemacher et al., 1965; Munro and Fleck, 1969). Purified calf liver RNA was used as standard and samples extracted for DNA were compared with purified calf thymus DNA. As standards, bovine serum albumin, RNA and DNA obtained from Sigma (St. Louis) were used. The different parameters studied were subjected to statistical analysis by Student’s t-test. Results and Discussion
Biochemical
procedures
Livers from the same animals were quickly excised, frozen in liquid nitrogen and stored at -20°C till analyzed for total proteins and nucleic acids (RNA and DNA). Total proteins were estimated by the modified Lowry method of Schacterle and Pollack (1973). Bovine serum albumin was used as a standard. Nucleic acids
The administration of (+)-usnic acid was found to decrease the proliferation of polychromatic erythrocytes at 24 and 48 h of exposure at both doses (100 and 200 mg/kg). However, the depletion was statistically significant (P c 0.001) at the highest dose only after an exposure of 24 h (Table 1).
TABLE 2 EFFECT OF (+)-USNIC ACID ON PROTEIN AND NUCLEIC ACID CONTENTS OF LIVER IN MICE Oral treatment (dose) Distilled water (IO ml/kg) Usnic acid t loo mdkg) Usnic acid t200 mg/kg)
Protein (mg/lOO mg)
RNA WI00
DNA (lrg/lOfJ mg)
mg)
+24 h
+48 h
+72 h
+24
h
+48 h
+72 h
+24 h
+48
h
+72 h
12.2 l 2.2 12.1 l 1.3 12.4 f I.9
II.8 zt I.8 9.4 f 0.5 9.4 l I.3
II.9 l 1.2 12.3 l I.0 I I.6 * I.8
599 l 59 503 f 48 487 f 70
616 f 52 569 f 63 599 * 73
615 zt 65 625 * 73 609 zt 62
308 l 30 321 f 32 315 l 6t3
312 f 45 325 *40 311 * 49
315 l 34 310 f 42 319 f 38
Tabular values represent the mean f S.E.M. of 5
mice/group.
219
TABLE EFFECT
3 OF (+)-USNIC
Oral treatment
ACID
ON THE
FREQUENCY
+24 h
OF MICEONUCLEI
IN FEMORAL
CELLS
OF MICE
+72 h
+48 h
(dose) Polychromatic cells screened
Micronucleated
Polychromatic
Micronucleated
Polychromatic
Micronucleated
polychromatic
cells screened
polychromatic
cells screened
polychromatic
(N)
(‘%I)
(N)
(‘%I)
(N)
(‘X)
Distilled water (IO ml/kg)
6560
0.28
f
0.05
5051
0.22
f
0.09
4518
0.22
f
0.02
Usnic acid
4500
0.37
& 0.10
4667
0.57
*
0.15
4943
0.33
*
0.05
4606
0.48
+ 0.09
4001
0.41
f
0.07
4448
0.39
l
0.12
(100 mg/kg) Usnic acid (200 mgkg) Percentile values represent the mean
f
S.E.M.
of 5 mice/group.
The results on the quantification of proteins nucleic acid levels of liver cells in the same animals showed an apparent reduction in the levels of RNA at 24 and 48 h. Proteins appeared to be reduced at 48 h only for both doses (Table 2). Although the depletions were all statistically insignificant, the effect was consistent enough for the authors to consider that (+)-usnic acid may block the translation process by interferring with RNA biosynthesis (Tata, 1974; Brawerman, 1974; Lone et al., 1986; Al-Ahdal et al., 1988). The observed mitodepressant effect on the polychromatic erythrocytes may be due to an effect on mRNA molecules. Although comparable studies on (+)-usnic acid are scanty in literature, our results are in agreement with the findings on (-)usnic acid which was shown to be cytotoxic in some in vitro tests (Takai et al., 1979; Huovinen and Lampero, 1988). The effect of (+)-usnic acid on clastogenic activity was also studied using the femur cells of mice. There was an apparent increase in the micronucleated polychromatic erythrocytes at 24 and 48 h for both doses (Table 3). However, the increases were statistically insignificant and values returned to normal at 72 h. Simultaneous observations on DNA levels in liver cells showed no significant changes between the control and treated groups (Table 2). The increase in micronucleated polychromatic erythrocytes without affecting the level of DNA suggests a toxic effect of (+)-usnic
acid on the spindle apparatus (Schmid, 1975). Further work on the anti-cancer!cytotoxic potential of (+)-usnic acid and the clastogenicity of the two isomers is required to elucidate the exact mode of action of these compounds. Acknowledgement
We are thankful to Dr. A.V.N.A. Rao for his kind remarks and suggestions, References Al-Ahdal.
M.N..
Cytotoxicity
McGarry.
T.J.
and
Hannan.
M.A.
(1988)
of khat (Cutha edulis) extract on cultured mam-
malian cells: Effects on macromolecule
biosynthesis.
Mu/u-
tion Reseurch 204. 621-642. Brawerman.
G.
(1974)
Eukaryotic
Review cf Biochemistry Hayashi.
massenger
RNA.
Annual
43. 62 1-642.
M.. Sofuni. T. and Ishidate. M.. Jr. (1984) A pilot ex-
periment
for the micronucleus
multidose levels method. Huovinen,
test. The multi-sampling
Mutution
K. (1988) The content of protocetraric
ferent decoctions of Cetruriu islundicu. 36th on Mrdicinul
at
Reseurch 141, 165-169.
Plunt Reseurch (Freiburg,
acid in dif-
Annwl
12-16
Congress
September),
p. 31, (P2--12). Huovinen,
K. and Lampero.
inhibitor
M. (1988) Usnic acid as a mitotic
in the Album test. 361h Annuul Congress on Medici-
nal Plum
Research
(Freiburg.
12-16
September),
p. 31
and Basu. B.D. (1984) lncliun Medicinul
Plunrs.
(P2--12). Kirtikar.
K.R.
Lalit Mohan
Basu Pub.. Allahabad.
India. pp. 276&2761.
Kupchan. S.M. and Kopperman. H.L. (1975) Usnic acid: Tumor inhibitor isolated from lichen. Esperientiu 31. 625-626.
220 Lone, K.P., Chaudry, M.A. and Matty, A.J. (1986) Effect of thyroid hormone and actinomycin-D on protein and nucleic acids metabolism in carp liver and muscle. Pakistan Journal of Zoology 18, 297-309.
Munro, H.N. and Fleck, A. (1969) Analysis of tissue and body for nitrogenous constituents. In: H.N. Munro (Ed.), Mammalian Protein Metabolism. Vol. 3, Academic Press, London, pp. 423-525. Schacterle, G.R. and Pollack, R.L. (1973) A simplified method for quantitative assay for small amounts of proteius in biological materials. Analyrical Biochemistry 51, 654-655. Schmid, W. (1975) The micronucleus test. Mutation Research 31, 9-15.
Shibamoto, T. and Wei, C.I. (1984) Mutagenicity of lichen constituents. Environmental Mutagenesis 6, 757-762. Shibata, S. (1978) Lichen Substances. In: F. Korte and M.
Goto (Eds.), Natural Compounds. Georg Thieme Pub., Stuttgart, pp. 208-223. Takai, M.. Uehara, Y. and Beisler, J.A. (1979) Usnic acid derivatives as potential antineoplastic agents. Journal of Medicinal Chemistry 22. 1380-l 384. Tata, J.R. (1974) Growth and developmental action of thyroid hormones by actinocycin-D and puromycin. Nature 197. 1167-l 168. Trease, G.E. and Evans, WC. (1978) Pharmacognosy. Bailliere Tindall, London, pp. 621-623. Wannemacher, R.W., Jr., Bank, W.L.. Jr. and Wunner. W.H. (1965) Use of single tissue extract to determine cellular protein and nucleic acids concentration and rate of amino acid incorporation. Analytical Biochemistry I I, 320-326. Windholz, M. and Budavair, S. (1983) The Merck Index. 10th Edn. Merck and Co. Inc., Rahway NJ, p. 1414.