Role of prostaglandins in bone metabolism - PubMed Central Canada

Journal of the Royal Society of Medicine Volume 72 January 1979

Role of prostaglandins in bone metabolism:

27

a review

D Atkins2 PhD M Greaves2 MB MRCP K J Ibbotson BSC T J Martin2 MD FRACP Department of Chemical Pathology, University of Sheffield Medical School,

Sheffield SJO 2RX

Hypercalcaemia is a frequent complication of malignant disease in man, but the pathophysiology of bone destruction in cancer is poorly understood. Since the development of specific and sensitive radioimmunoassays for parathyroid hormone (PTH), there have been a few well-documented reports of PTH production by nonendocrine tumours (Knill-Jones et al. 1970, Greenberg et al. 1973). However, most patients with hypercalcaemia and nonmetastatic cancer have normal or suppressed PTH levels (Powell et at. 1973). Thus, although 'ectopic' PTH production can occur, it may only very rarely be the cause of hypercalcaemia in cancer, and other factors must be sought. Over the last few years several agents have been proposed as a cause of bone destruction in cancer (Table 1), and major current interest centres on the role of Table 1. Some causes ofhypercalcaemia in human cancer Causative agent

Tumour

Reference

Parathyroid hormone

Renal cell carcinoma Hepatoma Breast

Greenberg et al. (1973) Buckle et al. (1970) Gordan et al. (1966) Mundy, Luben & Raisz (1974) Mundy, Raisz et al. (1974) Bennett et al. (1975) Dowsett et al. (1976)

Osteolytic sterol Osteoclast activating factor (OAF) Prostaglandins

Myeloma Leukaemia Breast

the prostaglandins (PGs). Chase & Aurbach (1970) first showed that PGF2z, stimulated bone cyclic adenosine monophosphate (AMP) production whilst Klein & Raisz (1970) showed that PGs were potent bone resorbing agents in tissue culture. Evidence for a role of PGs in the hypercalcaemia of cancer came from the work of Tashjian et al. (1972) who found that a mouse fibrosarcoma in tissue culture produced a bone resorbing agent which could be identified as PGE2. The purpose of this present paper is to review some of the current hypotheses concerning the role of PGs in the physiology and pathophysiology of bone resorption.

Hypercalcaemic cancer in experimental animals Most of the information concerning the production of PGs as bone resorbing agents by cancers has come from animals bearing hypercalcaemia-producing tumours such as the HSDM1 fibrosarcoma in mice, Walker tumour in rats and the VX2 carcinoma in rabbits. The mouse fibrosarcoma produces hypercalcaemia about 21 weeks after implantation (Tashjian et al. 1972), and this can be prevented by treatment of the animals with indomethacin (a PG synthetase inhibitor) (Tashjian et al. 1972) or hydrocortisone (Tashjian, Voekkel & Levine Paper read to Section of Endocrinology, 25 May 1977. Accepted 8 November 1977 2 Present addresses: D Atkins, Department of Biochemistry, University of Hong Kong; M Greaves, Department of Medicine, University of Sydney, Australia; T J Martin, Department of Medicine, Repatriation General Hospital, University of Melbourne, Australia 0 1 41-0768/79/010027-08/$O 1.00/0

00 1979 The

Royal Society of Medicine


28

1977). Moreover, when tumour cells were grown in monolayer culture, the supernatant medium was found to contain bone resorbing activity which was not present when cells were grown in the presence of indomethacin (Tashjian et al. 1972). Essentially similar studies have been performed using the VX2 carcinoma (Tashjian et al. 1973) and the Walker tumour in rats (Powles et al. 1973) leading to the conclusion that PGE2 is the major cause of increased bone destruction by these tumours. In spite of this evidence it has been difficult to show that circulating levels of PGE2 are increased in malignant hypercalcaemia (Tashjian et al. 1972, 1973), and intravenous infusion of relatively high concentrations of PGE2 does not produce hypercalcaemia (Beliel et al. 1973, Robinson & Parsons 1975). However, PGE2 is rapidly cleared from the circulation and converted to 13,14-dihydro- 1 5-keto PGE2 (Figure 1) and Tashjian, Voekkel & Levine (1977), 15-hydroxyprostaglandin

O

"\zN,-< ~ OH

HO

O

dehydrogenase COOH PCO HO

PGE2 Reduction

&I3double 1n-oxidation o

o ,)%. \/COOH

HO

of bond

COOH 0 Major urinary metabolite

~

.N

w-oxidation

COOH

0

HO

15-keto-dihydro-PGE2

Figure 1. The metabolism of PGE2 in man

using the mouse fibrosarcoma model, have found that plasma levels of this metabolite were elevated some 10-fold before hypercalcaemia developed, whilst PGE2 levels were only minimally increased. It therefore seems likely that the rate of production of PGE2 is rather more important than the absolute plasma levels reached, hence constantly exposing the bone cells to increased amounts of PGE2. An alternative view would be that marked hypercalcaemia only develops once tumour cells have invaded bone. The VX2 carcinoma presented a suitable model to test this hypothesis since, when cells are injected intramuscularly, tumour deposits can be found in bone after a latent period. Indomethacin readily prevents the development of hypercalcaemia (Tashjian et al. 1973). Galasko & Bennett (1976) have shown that when the tumour was injected into the bone marrow there was a rapid increase in the number of osteoclasts (bone resorbing cells) near the site of tumour injection and, furthermore, that when rabbits were treated with indomethacin the increase in osteoclast count following tumour injection was less. Thus, it is possible to propose a model whereby a PG, probably PGE2, can be responsible for bony destruction in both primary and metastatic disease. If PG production by the primary tumour is markedly increased, although plasma levels are only slightly elevated, this increase will be sufficient to cause a small increase in bone resorption, possibly due to osteoclast activation. This will cause the production of microscopic resorption cavities in bone which will represent suitable sites for the development of bony metastases. Once tumour cells have spread to bone then the large local increase in PG levels will stimulate further increases in bone destruction leading to the development of gross hypercalcaemia (Figure 2). Clinical models Direct evidence that PGE2 is a cause of hypercalcaemia in man is lacking despite the size of the literature. There is little convincing data to show that plasma levels of PGE2 are markedly

Journal of the Royal Society of Medicine Volume 72 January 1979 TUMOUR

29

HISTOLOGICAL PATTERN

BONE

PRE-NUASTATIC

Local erosion od trabeculae

iCa

, Urine

-

Smaill increase in PGUs -_

Parathyroid shutdoawn IQ compensated

local erosion.

METASTATIC

Tumour cello Bony metastases - locally produced PG:s.

T ca

Deposits in bone -

-

a lercakaemi

Large increase in resorption

Kidney unable to deal with increased filtered load ..

HYPERCALCAEMIA

Figure 2. Proposed model to show how hypercalcaemia may develop in premetastatic and metastatic cancer

increased in hypercalcaemic cancer (Robertson et al. 1975, Demers et al. 1977), but there are data to show that the urinary excretion of PGE-M is increased in hypercalcaemic cancer (Seyberth et al. 1975). However, attempts to treat the hypercalcaemia with indomethacin have not fulfilled their promise (Coombes et al. 1976). Nevertheless, it is important to be able to predict those patients who, although normocalcaemic at surgery, may later develop bony metastases. Powles et al. (1973) showed that when fragments of human breast carcinomata were cocultured with bone, resorption occurred. Inclusion of indomethacin in culture media could prevent the resorption and, on the basis of radioimmunoassay studies, it was later suggested that the bone resorption was due to PGE2 (Dowsett et al. 1976). Bennett et al. (1975) found that when PG production by breast tumours in short-term culture was high, the same patients subsequently were found to develop bony metastases. Bennett et al. (1975) made the suggestion that the bone resorbing factor was PGF2,, although this appears unlikely in view of the relative low bone resorbing activity of PGF2, (Dietrich et al. 1975). We have carried out similar studies but our data suggest that the incidence of production of bone resorbing factors by breast tumours is lower than that suggested by Powles et al. (1973). However, all such studies suggest a potential value of such systems in evaluating the potential production of bone resorbing agents by tumours. Coincubation studies of fragments of human renal cortical carcinoma (hypernephroma) tissue with bone have been performed. This tumour has been reported to be associated with hypercalcaemia and with the ectopic production of PTH (Greenberg et al. 1973). The cells of the tumour have ultrastructural similarities to cells of normal proximal or distal tubules (Hunt et al. 1978), and it is possible at surgery to obtain control unaffected renal tissue. It has been shown that there is an increase in adenylate cyclase activity in tumour cell membranes compared to control tissue (Hunt et al. 1978) and this is reflected by a higher level of cyclic AMP in the renal vein than artery in the patients at surgery. It was suggested that this derangement of adenylate cyclase could be concerned with metabolic or differentiated functions of the tumour cells. Because of the association between this tumour and hypercalcaemia, the potential production of bone resorbing factors by this tumour was investigated.


30

Using the coculture system we have shown that a number of renal cortical carcinomata do produce a bone resorbing factor which by a variety of criteria can be identified as a prostaglandin (probably PGE2) (Atkins, Ibbotson et al. 1977). A typical case is shown in Figure 3, where it can be seen that the tumour explants caused bone resorption which was 40 30-

E

-1

I

I ._

o lo 0

-jCon

Tumn

Tum IDM

Tum. Norm PTH Theo Kid

Figure 3. Bone resorption by human renal cortical carcinoma in tissue culture. Fragments of tumour were cocultured with mouse calvaria for 3 days. PGE levels were estimated in pooled culture media. (IDM, 14 ,mol indomethacin, Theo, I mmol theophylline)

inhibited when indomethacin was included in the culture medium. Inclusion of theophylline (a cyclic AMP phosphodiesterase inhibitor) led to an enhancement of bone resorption. Direct measurement of PGE by radioimmunoassay helps confirm that the bone resorbing factor is indeed PGE. Studies using theophylline provide a clue to the possible significance of the increased adenylate cyclase activity. It is likely that the increased rate of cyclic AMP production may act to stimulate the synthesis of PGs which in turn could have a variety of metabolic effects on bone resorption. Furthermore, it is possible that, because PGE2 itself can stimulate adenylate cyclase, the system is self-perpetuating, that is, there is a continued drive to produce PGE2 (Figure 4). Such possibilities have been investigated by incubation of fragments of normal rat kidney with '4C-arachidonic acid and isolating the PGs formed. Table 2 shows that both theophylline and dibutyryl cyclic AMP enhance the metabolism of arachidonic acid to PGs. What we cannot define from these studies is whether bone resorption is the specific function of the PG production by the renal tumour cells. It is quite possible that increased adenylate cyclase activity and PG production may be associated with disturbances in the production of other hormones such as erythropoietin (Greaves et al. 1977) or ectopic hormones known to be occasionally produced by renal cortical carcinomata, or indeed with some other metabolic function. It has been shown clinically that hypercalcaemia in some patients with renal cortical carcinoma may well be related to excessive PG production (Brereton et al. 1976, Robertson et al. 1975).


31

CONTROL OF PROSTAGLANDIN PRODUCTION

CEFFECTS

Figure 4. Possible mechanism whereby cyclic AMP controls PGE2 synthesis in renal cortical carcinoma

The role of prostaglandins in normal bone metabolism The fact that PGs may cause the hypercalcaemia in cancer strongly suggests that they play a part in normal bone metabolism, but this aspect of bone physiology has received little attention. It is accepted that PGE2 is the most potent bone resorbing PG in tissue culture (Dietrich et al. 1975) and that, in several respects, its action is similar to that of PTH in that it stimulates cyclic AMP production (Chase & Aurbach 1970) and inhibits collagen synthesis (Raisz & Koolemans-Beynen 1974). Since PGs usually act relatively close to their site of action it is essential that, if they are to play an important role in bone metabolism, they should be produced locally by bone cells. This possibility has not been investigated. In an attempt to investigate further the effects of PGs on bone and bone cells we have used cells from a 32P-induced transplantable osteogenic sarcoma in the rat. These tumours and their cells have a pattern of hormonal responsiveness which is very similar to that of normal bone cells (Martin et al. 1976, Atkins, Hunt et al. 1978). In particular, the ability of various PGs to stimulate cyclic AMP formation (Atkins & Martin 1977) exactly parallels their in vitro bone resorbing activity (Tashjian, Tice & Sides 1977, Raisz et al. 1977). Furthermore, these hormonally responsive cells offer a unique opportunity to study the actions of other short-lived prostaglandin-like substances which would be almost impossible to test in more conventional bone systems. Prostaglandin biosynthesis and metabolism is very complex. The biosynthesis pathways are outlined in Figure 4. Briefly, after the formation of an obligatory cyclic endoperoxide Table 2. Synthesis ofprostaglandins from 4C-arachidonic acid. (Smallfragments were incubatedfor I hour in the presence of l4C-arachidonic acid, adrenaline and reduced glutathione. After extraction with ethyl acetate, PGs were chromatographed on silicic acid (Jaffe & Parker 1972)) ng/mg protein/h (% control)

Control Cu2 + 2 x 10-5 mol/l Indomethacin 2 x 10-3 mol/l Theophylline 2 x 10-3 mol/l Dibutyryl cyclic AMP 1 x 10-3 mol/l

PGA

PGE

PGF,,

Total

0.89 1.14 0.29 0.74 1.38

0.18 (100%) 0.90 (500%) 0.17 (94%) 0.36 (200%) 1.29 (717%)

0.22 (100%) 2.10 (9540%) 0.13 (59%) 0.86 (404%) 2.72 (1236%)

1.29 4.14 0.59 1.96 5.39

(100%) (128%) (36%) (83%)

(155%)

(100%) (321 %) (46%) (152%) (417%)

32


intermediate PG biosynthesis can follow one or more alternative pathways. First, there is the formation of biologically inactive hydroxyacids. Secondly, the endoperoxides can be converted to almost any of the conventional PGs all with somewhat varying biological activities, or to short-lived compounds-thromboxane A2 and prostacyclin (PGI2). The role of these compounds is only understood well in the blood platelet. Thromboxane A2 is a potent aggregatory agent produced by the platelet, whilst prostacyclin is a very potent antiaggregatory agent produced in the arterial walls (Hamberg et al. 1975, Gryglewski et al. 1976). Table 3. Effect of PG endoperoxide analogues on bone resorption in culture and on cyclic AMP production in isolated osteogenic sarcoma cells

U44069 10-9mol/ 10- 8mol/I 10-7 mol/1 10-6 mol/1 10-5 mol/1 U46619 10-9mol/1 10-8 mol/I

10-7mol/I 10-6 mol/ 10-5 mol/l Control

(% 45Ca released)

Bone resorbing activity@

Cyclic AMP production (pmol/106 cells/10 min)

19.7 +0.9 19.9 + 1.1 21.5 + 1.4 24.1 + 1.6 27.1 + 1.5 21.3+ 1.5 20.7+ 1.5 20.5+ 1.3 22.4+ 1.0 23.4+ 1.9 20.2 +0.7

6.57 +0.68 7.05 + 0.53 15.44+ 1.14 18.72+2.53 52.05 +4.43 7.15+0.16 10.07+ 1.34 14.02+0.82 19.23+ 1.84 33.38 + 2.79 6.26 +0.15

* Unpublished data of Dr L G Raisz. For details of the bone resorption system

see Dietrich et al. (1975)

Many other tissues produce thromboxane A2 and prostacyclin, which may represent the true local regulatory PGs. The cyclic endoperoxides themselves will stimulate platelet aggregation (Hamberg et al. 1975). In conventional bone culture systems there is no convincing evidence for a bone resorbing effect (Raisz et al. 1977). However, due to their relative instability it is generally necessary to use stable analogues of these compounds. Two such analogues are known (containing an epoxymethano linkage, U44069 & U46619). These analogues do not have significant bone resorbing effects and are only weak stimulators of cyclic AMP formation in osteogenic sarcoma cells (Table 3). It has not been possible to test the effect of prostacyclin in any bone system, but it might be predicted that it would be a potent stimulator of cyclic AMP production in the osteogenic sarcoma cells. None of the known major PGE2 metabolites are as effective stimulators of bone resorption in tissue culture as PGE2 (Tashjian, Tice & Sides 1977, Raisz et al. 1977), nor do they, except at high doses, stimulate cyclic AMP formation in osteogenic sarcoma cells (Figure 5) (Atkins & Martin 1977). These data allow the inference that, although it is easy to demonstrate an increase in circulating levels of PG metabolites in hypercalcaemic cancer, the bone resorption occurring is due either to PGE2 itself or to a shortlived PG intermediate such as thromboxane A2 or prostacyclin. Several groups of workers have suggested that the action of thyroid stimulating hormone (TSH) is via a stimulation of PGE2 synthesis in thyroid membranes and that, in turn, PGE2 stimulates cyclic AMP production and hence thyroid secretion (Sato et al. 1972). We have evidence to suggest that PGE2 is not involved in the action of PTH on osteogenic sarcoma cells. During the enzymic dispersion of isolated cells there is a selective loss of responsiveness to PTH but not to PGE (Martin et al. 1977). Preincubation of cells with PG biosynthesis inhibitors (aspirin or indomethacin) does not impair subsequent responsiveness to PTH, neither does preincubation with arachidonic acid enhance hormone responsiveness. A further line of


33

ARACHIDONIC ACID

I,

CYCLIC ENDOPEROXIDES

THROMBOXANE

A2

THROMBOXANE B2

PROSTACYCLIN IPGX)

6-KETO

PGFla

PROSTAGLANDINS

HYDROXYACIDS

PG METABOLITES

Figure 5. Pathways for the biosynthesis of prostaglandins from arachidonic acid

evidence is that PG antagonists (SC- 19220 and 7-oxa- 1 3-prostynoic acid) selectively antagonize the action of PGE2 (Waller et al. 1979). Thus it is possible to suggest that the action of PGE2 (or other PG-like substances) is independent of PTH action, at least in these tumour cells which are closely related to bone. It seems very unlikely that PG effects on bone are mediated by peripherally-produced PGs and it is necessary to consider whether PGs can play a local role in the regulation of bone turnover. This role is particularly hard to define, but remembering the local regulatory effects of PGs in the platelet, it is possible that a PG plays an important role in the minute-to-minute regulation of bone turnover. In the absence of any apparent stimulation, for example in hypoparathyroidism, there is a remarkable degree of self-regulation in bony tissue - a possible role for prostaglandins?

Conclusions Although there is ample evidence that PGs are important in the pathogenesis of hypercalcaemia (bone destruction) in cancer, the role of PGs in normal bone metabolism is poorly understood. However, it is anticipated that PGs may play a central role in the control of bone turnover and possibly in the minute-to-minute regulation of plasma calcium.

Acknowledgment: We are grateful to Dr L G Raisz, University of Connecticut, for allowing us to use his unpublished data.

References Atkins D, Hunt N H, Ingleton P M & Martin T J (1977) Endocrinology 101, 555 Atkins D, Ibbotson K J, Hillier K, Hunt N H, Hammonds J C & Martin T J (1977) British Journal of Cancer 36, 601 Atkins D & Martin T J (1977) Prostaglandins 13, 561 Beliel 0 M, Singer F R & Coburn J W (1973) Prostaglandins 3, 237 Bennett A, McDonald A M, Simpson J S & Stamford I F (1975) Lancet i, 1218 Brereton H D, Halushka P V, Alexander R W, Maron D M, Kreiser H R & DeVita V T (1976) New England Journal of Medicine 291, 83 Buckle R M, McMillan M & Mallinson C (1970) British Medical Journal iv, 724 Chase L R, Aurbach G D (1970) Journal ofBiological Chemistry 245, 1520 Coombes R C, Neville A M, Bondy P K & Powles T J (1976) Prostaglandins 12, 1027 Demers L M, Allegra J C, Harvey H A, Lipton A, Luderer J R, Martel R & Brenner D C (1977) Cancer 39, 1559 Dietrich J W, Goodson J M & Raisz L G (1975) Prostaglandins 10, 231 Dowsett M, Easty G C, Powles T J, Easty D M & Neville A M (1 976) Prostaglandins 11, 447 Galasko C S B & Bennett A (1976) Nature 263, 508 Gordan G S, Satino T J, Erhardt L, Hansen J & Labich W (1966) Science 151, 1226 Greaves M, Martin T J, Atkins D, Gyde O H B & Harris B R (1977) Journal of Endocrinology 75, 210 Greenberg P B, Martin T J & Sutcliffe H S (1973) Clinical Science 45, 183

34


Gryglewski R J, Bunting S, Moncada S, Flower R J & Vane J R (1976) Prostaglandins 12, 685 Hamberg M, Svensson J & Samuelsson B (1975) Proceedings of the National Academy of Sciences of the USA 72, 2994 Hunt N H, Shortland J R, Michelangeli V P, Hammonds J C, Atkins D & Martin T J (1978) Cancer Research 38, 23 Jaffe B M & Parker C W (1972) In: Infertility Control. WHO, Stockholm; p 69 Klein D C & Raisz L G (1970) Endocrinology 86, 436 Knill-Jones R P, Buckle R M, Parsons V, Calne R Y & Williams R (1970) New England Journal of Medicine 282, 704 Martin T J, Crawford A, Coulton L, Atkins D, Hunt N H, Dawborn J K, Ingleton P M & Underwood J C E (1977) Proceedings of the 6th Parathyroid Conference. Excerpta Medica, Amsterdam; p 262 Martin T J, Ingleton P M, Underwood J C E, Michelangeli V P, Hunt N H & Melick R A (1976) Nature (London) 260, 436 Mundy G R, Luben R A & Raisz L G (1974) New England Journal of Medicine 290, 867 Mundy G R, Raisz L G, Cooper R A, Schechler G P & Salmon S E (1974) New England Journal of Medicine 291, 1041 Powell D, Singer F R, Murray T M, Minkin C & Potts J T jr (1973) New England Journal of Medicine 289, 176 Powles T J, Clark S A, Easty D M, Easty G C & Neville A M (1973) British Journal of Cancer 28, 316 Raisz L G, Dietrich J W, Seyberth H W, Hubbard W & Oates J A (1977) Nature 267, 532 Raisz L G & Koolemans-Beynen A R (1974) Prostaglandins 8, 374 Robertson R P, Baylink D J, Marini J J & Adkinson H W (1975) Journal of Clinical Endocrinology and Metabolism 41, 164 Robinson C J & Parsons J A (1975) Journal of Endocrinology 64, 14P Sato S, Szabo M, Kervalski K & Burke G (1972) Endocrinology 90, 343 Seyberth H W, Segre G V, Morgan J L, Sweetman B J, Potts J T & Oates J A (1975) New England Journal of Medicine 293, 1278 Tashjian A H jr, Tice J E & Sides K (1977) Nature (London) 266, 645 Tahsjian A H jr, Voekkel E F & Levine L (1977) Biochemical and Biophysical Research Communications 74, 199 Tashjian A H jr, Voelkel E F, Levine L & Goldhaber P (1972) Journal ofExperimental Medicine 136, 1329 Tashjian A H jr, Voelkel E F, Levine L & Goldhaber P (1973) Prostaglandins 3, 515 Waller P C, Atkins D & Martin T J (1979) Clinical and Experimental Physiology and Pharmacology (in press)

Conferences 2-4 February 1979 Fifth Glasgow temporal bone dissection course Information from: The 1880 Foundation, c/o Ear, Nose and Throat Hospital, 306 St Vincent Street, Glasgow G2 5RX

4 February 1979 Societe Nationale Francaise de Proctologie Meeting in Paris: Les constipations terminales ou dischesies anorectales Information from: Dr E Parnaud, 62 Boulevard Malesherbes, 75008 Paris 22-24 February 1979 Deuxieme congres FranVais des services d'aide medicale urgente to be held in Toulouse Information from: Docteur Jean-Pierre Gaston, SAMU, Centre Hospitalier Universitaire de Toulouse, 31052 Toulouse Cedex, France 4-7 June 1979 Second international clinical genetics seminar to be held in Athens Information from: 2nd Department of Pediatrics, University of Athens, Aglaia Kyriakoy Children's Hospital, Athens, Greece

2-7 September 1979 Tenth international conference on health education to be held in London Information (in English, French or Spanish) from: The Conference Centre, 43 Charles Street, London WIX 7PB, England 20 October 1979 Societe Nationale Fran,aise de Proctologie Meeting in Marseille: Les incontinences anales Information from: Dr P Delecourt, H6pital Nord, Marseille December 1979 Ninth meeting of the international study group for steroid hormones to be held in Rome Information from: Professor Carlo Conti, Istituto di Patologia Medica, Policlinico Umberto I, Universita di Roma, Rome, Italy

4-10 December 1979 Fourth biennial meeting of the Asian Federation for Organizations for Cancer Research and Control (AFOCC) hosted by the Indian Cancer Society in Bombay Teaching programme in cancer chemotherapy will be conducted by the International Union Against Cancer (UICC), Geneva Information from: Organizing Secretaries, IV Conference of AFOCC, Tata Memorial Hospital, E Borges Marg, Parel, Bombay-400 012, India