[1] 234w The findings in the experimental neu- ropathy consisted of axonal swelling with thinning of the myelin sheath or denudation of myelin near the node of Ranvier associated with accumulated masses of neurofilaments. This type of abnormality is very similar to the giant axonal change described by Asbury et al. (4) in a sporadic case of a slow- ly progressive mixed polyneuropathy without known exposure to toxins. It is also of interest that. the peripheral nerve can show neurofilamentous alter- ations with other agents including acrylamide (5), /3,/3'-iminodipropionitrile (6), and vincristine (7) but none are identical to those described here with MBK or in the case of the giant axonal neuropathy. It is also important that in our experimental neuropathy focal areas of denudation of myelin were present without swelling of the axon. This change could be secondary to axonal damage, although we cannot rule out the possibility of direct toxic- ity of MBK for the myelln sheath.Safe atmospheric levels of MBK should be established; it is also neces- sary to ascertain whether the recom- mended threshold limit value of 100 ppm for MBK provides an adequate margin of safety for workers exposed to this solvent.Note added in proof: After this man- uscript was submitted for publication, Spencer and Schaumberg (8) reported similar findings in rats exposed to MBK at 1300 ppm, 6 hours per day, 5 days a week, for up to 4 months.

[1] 215w Serum from ten normal rabbits all showed heat-labile stimulation of bone resorption (Table 1) and only 1 of 16 individual assays failed to show a significant difference between experimental and control cultures after 6 days. The mean increase in release of 45Ca was approximately 60 percent and most of this occurred during the second 3 days in culture. When an increase in release of 45Ca was observed during the first 3 days, it was usually less than 30 per- cent. The experimental-to-control ratios achieved were not as high as those at- tained with other experimental models that employed parathyroid hormone or osteoclast-activating factor from human leukocytes (4, 5), largely because (i) no preculture was used to remove ex- changeable 45Ca and (ii) a relatively high rate of control resorption was produced by the presence of 50 percent heated serum in !the medium. At the end of 6 days, bones cultured in un- heated serum showed marked histologic change; compared with bones cultured in heated serum, there was loss of ma- trix and proliferation of osteoclasts and Table 1. Bone-resorbing activity of rabbit serum. Values are means ± standard errors for number of assays given in parentheses; each assay consisted of four pairs of bones cultured with the indicated serum. C6, the sixth component of complement.

[2] 78w Rabbit serum 6-day '5Ca release (experimental/control Experimental Control ratio) Normal, unheated Normal, heated* 1.59 ± 0.06t (16) C6-deficient, unheated C6-deficient, heated* 1.05 0.05 (14) C6-deficient plus human C6-deficient plus human C6, unheated C6, heated* 1.41 ± 0.llt (4) C6-deficient plus guinea C6-deficient plus guinea pig C6, unheated pig C6, heated* 1.44 0.15t (4) Normal plus human C6 Normal 1.05 ± 0.02 (3) Normal plus guinea pig C6 * 56'C, 30 minutes. t Significantly greater than-1.0, P < .05.

[3] 39w Table 2. Prostaglandin E (PGE) content of media after 6 days of culturing rabbit serums and with indomethacin (10-5M). Values are means + standard e indicated number of cultures, each containing four bones per milliliter of mediun 6-day 45Ca

[4] 72w Serum Cultures release (No.) (count/min per milliliter) Heated normal> 6 2280 ± 160 Unheated Normal 8 3390 ± 220-, Normal plus indomethacin 8 1860 ± 170 C6-deficient 7 1990 + 120 C6-deficient plus guinea pig C6 7 2970 ± 190t C6-deficient plus guinea pig C6 plus indomethacin 7 1760 ± 230 * 56°C, 30 minutes. t Significantly different fromn heated normal serum. P < .05. different from C6-deficient serum, P < .05.

[5] 245w fibroblasts but no extensive cell necro- sis. To identify the heat-labile activity as C, serum from rabbits deficient in the sixth component of C (C6) (6) was used. The mean ratio of the release of 4"Ca in cultures containing unheated C6-deficient serum was not significantly different from those containing heated C6-deficient serum (Table I ). Only 2 of 14 assays from nine rabbits showed significant resorption, and in these re- lease of 4;Ca was less than 30 percent above the control. Functionally purified human or guinea pig C6 (7) signifi- cantly restored the bone-resorbing ac- tivity to C6-deficient serum (Table 1). Human C6 added to normal rabbit serum had no stimulatory effect. Guin- ea pig C6 caused a small (9 to 14 percent) increase in release of 4"Ca when added alone or to nor Addition of C2 was withou The delayed stimulation tion by C-sufficient serum tem suggested that synthesis ator by the bone tissue itse involved. Since prostagla potent stimulators of bone which produce a slower res such agents as parathyroi( or active vitamin D met; and are synthesized in many possibility that they mediate( effect was examined next. In has been shown to inhibit pI synthesis (9). The additi( agent at 10-5M to bone ( hibited the resorbing activit) rabbit serum as well as tha ficient serum supplemente( with various (Table 2). Indomethacin did not block rrors for the the response to parathyroid hormone or exogenous prostaglandin E2 (PGE2) 4ediumn PGE (Fig. 1).

[6] 17w (nanograms Media from these cultures were as- per culture) sayed for prostaglandin E (PGE) con- 0.1 ±0.1

[7] 387w tent by a specific radioimmunoassay procedure (10). Bones cultured with 3.3 + 0.4t heated normal serum released little 1.0 0.6 PGE into the medium (Table 2). How- 0.8 ±0.5 ever, bones cultured with unheated nor- 4.0+± 1.2$ mal serum showed not only increased 0.6 + 0.5 release of 45Ca but also a marked in- crease in PGE in the medium. Indometh- t Significantly acin inhibited the synthesis and release of PGE into the culture medium as well as release of 4,Ca from the bone. The addition of C6 to C6-deficient serum ma serum. resulted in a fivefold increase in the *effect PGE content of the medium, associated of resorpwith a 50 percent increase in the re- in this sys-lease of 4`Ca. Pooled extracts from If mght be bones cultured in unheated normal igh serum, which showed the greatest re- ndins art sorption, contained detectable PGE but sponse than only about 10 percent of the amount d hormone in the culture medium. Prostaglandin E abolites (8) was not detectable in extracts of bones tissues, the cultured in heated or C6-deficient serum serum. When media from the first and Idomethacin second 3-day periods were compared domethaclain for PGE content, more than 80 percent oostagladis of the total was found in the medium nc ofthis in from ithe second period, indicating de- y of normal layed synthesis and release from the t of C6-de-bone. The concentrations of PGE in d with C6 the medium in the second period were as high as 4 X 10-8M. Since PGE pro- duced by the bones would be diluted upon entering the medium, the concen- trations at the bone resorbing sites were probably much higher. Nevertheless, the addition of this amount of PGE1 or PGE, can cause significant stimulation of resorption in cultured bone (8). The 45Ca release experimental-to-control ratios of 1.45 and 1.71 were obtained ,ect of indo-when PGE, at concentrations of 148) on bone 10 -8M and 0-7M, respectively, was aced by vari-added to the cultures. s. Columns These results demonstrate the involve- ,andard error ment of C in the resorption of bone of cultures. and, furthermore, attribute this effect to ental/control the enhanced synthesis of prostaglandin ntly greater by the bone. Activation of C compo- rol ratio not nents, at least through C6, was required ater than 1.0. for this response.

[1] 92w Complement-Dependent Stimulation of Prostaglandin Synthesis and Bone Resorption Abstract. Complement-sufficient heterologous serum induced prostaglandin synthesis and resultant resorption in cultures of fetal rat long bones. Bone re- sorption was enhanced with unheated normal rabbit serum as compared to heated serum or serum from rabbits lacking the sixth component of complement (C6). Addition of functionally purified C6 restored resorptive activity in C6-deficient serum. Concentrations of prostaglandin E were increased in thie culture media of bones incubated with complement-sufficient serum. The resorptive effects of active serum as well as the appearance inhibited by indomethacin.

[2] 21w The resorption of fetal bone in or- gan culture can be stimulated by the addition of serum to the medium (1).

[3] 106w Histological evidence for the role of complement (C) in the breakdown of cartilage matrix, impaired growth, and increased bone resorption in cultures of mouse and chick bone rudiments containing serum was obtained by Fell, Lachmann, Coombs, Dingle, and Weiss (2). The destructive effects on carti- lage probably required the presence of connective tissue cells (3). These investigators attributed the observed effects to immune activation of C, since antiserum to cell surface antigens or to bone tissue intensified the activity and depletion of C from serum or the use of C-deficient serum abrogated the response. Complement activation increased the release of lysosomal en- zymes in their cultures.

[4] 94w We have assessed the role of C in stimulation of bone resorption by serum, using a quantitative assay based on the release of previously incorporated radiocalcium from fetal rat bone in organ culture (4). Nineteen-day, fetal rat long bone shafts (radius and ulna) previously labeled with 45Ca, were cultured in modified BGJ medium; 50 percent rabbit serum served as the of prostaglandin E in the media were source of C. The bones were cultured for 6 days, with one medium change at 3 days. Paired bones were used for con- trol and experimental cultures.

[5] 102w Results of previous work have sug- gested that antibody to cell surface antigens activates C on the cell mem- brane and initiates cartilage breakdown (2). The rabbit serum we used probably contained "natural" antibodies to some cell surface component of the fetal bone explants since we found SCIENCE, VOL. 185 7r 1.61-EJ3 Without Indomethocin 3 With Indomethocin T I '.o 5 co 1.4 u I 1.3 w o 1.2 cr In 1-V I.0o-0.9 a a Parathyroid Hormone j.g/ml 790 a a a b a b Prostoglon-Unheoted C6 def. serum din E Serum +C6 10-6a H eoled / C6 def. Serum Serum -::

[6] 186w ,k hemagglutinating antibodies for rat erythrocytes at the serum concentrations used in our studies. Additional studies (11) have shown marked enhancement of C-dependent bone resorption by antibodies obtained by immunization of rabbits with rat erythrocytes or rat bone sonicates. However, non-tissue- related antigen-antibody complexes, which activate either the classical or alternate C pathways (12), were ineffec- tive in enhancing the release of 45Ca in cultures containing C-sufficient serum. Moreover, preparations con- taining the active complement fragments C3a and C5a did not enhance bone resorption (13), which is not surpris- ing in view of the C6 requirement for the effect. These findings all indicate that C activation must take place on a cell membrane. The mechanism by which C activation increases PGE synthesis is not known. Conceivably, alteration of the cell membrane by C activation might provide a signal for increased PGE synthesis. Alternatively, C activation on a membrane might result in the release of fatty acids from membrane phospho- lipids which could then serve as pre- cursors of prostaglandins. Fatty acids may also stimulate bone resorption directly but only at concentrations of 10-4M or higher (14).

[7] 99w Prostaglandins have been detected in inflamed gingival tissue and exudates as well as in supernatants of rheumatoid synovial cultures (15). The pres- ent studies indicating a relationship be- tween C activation and PGE synthesis could help explain these findings as well as the associated pathologic breakdown of adjacent bone in such disorders as rheumatoid arthritis and periodontal disease. LAWRENCE G. RAISZ* Departments of Pharmacology and Toxicology and Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 ANN L. SANDBERG Laboratory of Microbiology and Immulinology, National Institute of Dental Research, Bethesda, Maryland cerned with growth regulation.

[8] 116w Triptolide (1) and tripdiolide (2) have recently been characterized as the novel and highly active antileukemic principles of the plant Tripterygium wilfordii Hook (1). The compounds at 0.1 mg/kg show impressive life-prolonging effects (that is, T/C -230) in mice afflicted with the L-1210 lymphoid leukemia (2). Biological and chemical data are presented in support of the im- portance of intramolecular catalysis (by a neighboring hydroxy group on the opening of an cpoxide by nucleophiles) for the mode of action of the antileukemic triptolides. The hypothesis is discussed in light of earlier proposals that other plant-derived tumor inhibi- tors may act via selective alkylation of the thiol groups of key enzymes con- cerned with growth regulation (3)(4)(5)(6)(7).

[9] 97w The nuclear magnetic resonance (NMR) spectra of the antileukemic triptolides 1 and 2 display resonances at T 7.16 (doublet, J = 11 hertz, 14-OH, disappears upon D.,O addition) and T 6.50 (doublet of doublets, J = 11 hertz and J -1 hertz, 14-H, collapses 30 AUGUST 1974 with ethyl alcohol. Samples were then dis- solved in benzene-ethyl acetate (60 : 40) and chromatographically purified by the method of R. M. Zusman, B. V. Caldwell, L. Speroff [ Prostaglandins 2, 41 (1972)], which separates PGE from fatty acids, PGA, PGB, and PGF (prostaglandins A, B, and F, respectively).

[10] 190w After conversion of the PGE to PGB by base hydrolysis, the samples were assayed with a commercial antibody to PGB., according to the method of L. Levine and H. Van Vunakis [Biochem. Biophys. Res. Commun. 41, 1171 (1970)]. This assay measures both PGE1 and PGE2, but was expressed in terms of PGEequivalents uncorrected for recovery (recovery of PGE, averaged 55 percent). 11. A. L. Sandberg and L. G. Raisz, in prepara- tion. 12. A. L. Sandberg, A. G. Osler, H. S. Shin, B. Oliveira, J. Immunol. 104, 329 (1970); A. L. Sandberg and A. G. Osler, ibid. 107, 1268 (1971). 13. L. G. Raisz, R. A. Snyderman, S. E. Mergen- hagen, unpublished observations. 14. B. Samuelsson, E. Granstrom, K. Green, M. Hamberg, Ann. N.Y. Acad. Sci. 180, 138 (1971); P. H. Stern, Experientia 27, 1061 (1971). 15. J. M. Goodson, F. Dewhirst, A. Brunetti, J. Dent. Res. 52, 182 (1973) (special issue); D. R. Robinson, H. Smith, L. Levine, Arthritis Rheum. 16, 129 (1973). 16. Supported by PHS grants AM 05205 and DE 03556. * Present address: Department of Medicine, University of Connecticuit Health Center-, Farmington 06032. 22 March 1974

[11] 163w to a singlet upon D20 addition). The highly distinctive JHCOH coupling con- stant (11 hertz) is attributable to the rigid trans orientation of the coupled protons resulting from strong hydrogen bonding between the 1 4-hydroxyl and the 9,11-epoxide groups (8). Free rota- tion Jl1cO1 coupling constants are typi- cally 3 to 6 hertz (9). The co-occurring triptolide 3 ("triptonide") differs struc- turally from 1 solely at C-14, which bears a ketonic function rather than a f,-oriented hydroxyl. Accordingly, the NMR spectrum of 3 does not show the resonances attributable to the hydrogen-bonded hydroxy-epoxide system. Triptonide shows no antileukemic activity in doses up to 0.4 mg/kg. These facts led us to hypothesize that the 9,11-epoxy-14,8-hydroxy system is nec- essary for the antileukemic activity of the triptolides. Furthermore, intramole- cular catalysis by the 14-hydroxyl group may assist selective alkylation of biological macromolecules by the 9,11- epoxide. Subsequent testing of the minor variants 14-epitriptolide [4, with 791 Selective Alkylation: A Biomimetic Reaction of the Antileukemic Triptolides?

[12] 53w Abstract. The potent antileukemic plant principles triptolide and tripdiolide contain a characteristic hydrogen-bonded 9,11-epoxy-14,8-hydroxy system. They alkylate propanethiol in a process which involves opening of the epoxide function with neighboring hydroxyl assistance. The reaction may mimic the inhibition of tumor growth via selective alkylation of the thiol groups of key enzymes con- -