Safety of milk from cows treated with bovine somatotrophin

Parkinson’s disease, but not Alzheimer’s disease, Lewy body variant associated with mutant alleles at cytochrome P450 gene genetic association between mutant alleles of the cytochrome P450 (CYP2D6B) gene and Parkinson’s disease has been reported.’ We determined to test this association SIR-A

and to see whether the same association also occurred in the other disease in which Lewy bodies are a prominent feature, Alzheimer’s disease, Lewy body variant (ADLB).è We assessed the genotype CYP2D6B by PCR in necropsydiagnosed cases of Parkinson’s disease, Alzheimer’s disease, ADLB, and control cases, all from the Newcastle upon Tyne area of the UK. CYP2D6B genotypes were assessed in 17 cases of Alzheimer’s disease, 19 ADLB, 19 Parkinson’s disease, and 17 controls. Mutant alleles were found in 6% of cases of Alzheimer’s, 16% of ADLB, 37% of Parkinson’s cases, and 21% of controls. Cases of Parkinson’s disease had a higher mutant allele frequency than other groups (p<0’05, chisquare test). Our results confirm the remarkable association between mutant alleles at CYP2D6B and Parkinson’s disease but fail to show any association between CYP2D6B and ADLB (or Alzheimer’s disease). These data suggest that those who have a mutant P450 allele have about a two-fold increase in their risk of developing Parkinson’s disease. The simplest explanation of this increase in risk is that this enzyme is, at least partly, responsible for metabolising an exogenous toxin, exposure to which can cause Parkinson’s disease. Poor metabolisers are at greater risk because the toxin will stay in their circulation for a longer time and thus cause more damage. Eventually, an age-related decline in the nigrostriatal system will lead to clinical expression of this damage. However, the fact that ADLB does not share this genetic risk factor suggests that Lewy bodies themselves are not a direct product of the disease but are a more secondary phenomenon. Further evidence for this conclusion is given by our observation that the 2 individuals who had APP717 Val→Ile encoded ADLBdid not have mutant CYP2D6B alleles. In contrast, several groups have shown that Alzheimer’s disease and ADLB (which share plaques as a pathological feature) share the genetic association between the E4 allele and disease (ref 4 and May 7, p 1155). Together, these data suggest a relation between aetiology, pathology, and disease nomenclature as outlined in the figure. In this scheme, since plaque-bearing diseases share aetiologies, plaque formation is closer to the aetiology of the disease. Lewy bodies occur in diseases with different aetiologies and are, therefore, likely to be more distant in the pathogenetic cascade and represent the pathological response of damaged neurons. A similar

Figure: Relation between aetiology, pathology, nomenclature

and disease

argument too occur

can be made with respect to tangles, since these in diseases with different genetic aetiologies.5

Work in the authors’ laboratories is supported by grants from the NIA (ROI AG11871-01) and by private donations.

Rempfer, Richard Crook, Henry Houlden, Karen Duff, Mike Hutton, Gareth W Roberts, Ravi Raghavan, Robert Perry, Ron

John

Hardy

Suncoast Alzheimer’s Disease Laboratories, Departments of Psychiatry, Pharmacology, Neurology, and Biochemistry, University of South Florida, MDC14, Tampa, FL 33613, USA; SmithKline Beecham Pharmaceuticals, The Pinnacles, Essex, UK; Department of Neuropathology, Newcastle General Hospital, Newcastle upon Tyne, UK; and Dementia Research Group, Departments of Neurology and Biochemistry, St Mary’s Hospital Medical School, London, UK

1

Smith gene

2

3

CAD, Gough AC, Leigh PN, et al. Debrisoquine hydroxylase polymorphism and susceptibility to Parkinson’s disease. Lancet

1992; 339: 1375-77. Perry RH, Irving D, Blessed G, Fairbairn AF, Perry EK. Senile dementia of Lewy body type: a clinically and neuropathologically distinct form of Lewy body dementia in the elderly. J Neurol Sci 1990; 95: 119-39. Lantos PL, Ovenstone IMK, Johnson J, Clelland CA, Roques P, Rossor MN. Lewy bodies in the brain of two members in the 717 (Valu to Ile) mutation of the amyloid precursor protein. Neurosci Lett

1994; 172: 77-79. 4

5

Corder EH, Saunders AM, Strittmatter WJ, et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993; 261: 921-23. Dlouhy SR, Hsiao K, Farlow MR, et al. Linkage of the Indiana kindred of Gertsmann-Straussler-Scheinker disease with neurofibrillary tangles. Nat Genet 1992; 1: 64-67.

Safety of milk from somatotrophin

cows

treated with bovine

and colleagues (July 16, p 197) express that the safety of milk from cows given bovine somatotrophin (BST) had not been established with adequate scientific rigour. They draw attention to the fact BST-milk contained that substantially increased concentrations of insulin-like growth factor-1 (IGF-1), which could adversely affect consumers in the long-term. They believe that toxicological tests with IGF-1 are inadequate because these tests only assess acute effects on gross organ indices, and suggest that more specific tests on gut cytokinetics and enzyme expression are indicated. In this laboratory we have examined the influence of recombinant human IGF-1 (rhIGF-1) on epithelial crypt cell proliferation in cultured explants of the adult human duodenal mucosa, with a stathmokinetic technique and crypt microdissection.’ Endoscopic duodenal biopsy was done in 6 men and 2 women (mean age 57 years) who were being routinely investigated for upper gastrointestinal disorders; biopsy specimens were histologically normal. Control and test specimens were cultured in a serum-free medium under identical conditions with separate culture dishes, and 400 ng/mL rhIGF-1 (Kabi Pharmacia) was added to test explants at the beginning of each experiment. Specimens were maintained in culture for 22 h before addition of 0-7 )ig/mL of vincristine sulphate to start the stathmokinetic experiments. They were cultured for a further 3 h, fixed in Carnoy’s fluid for 4 h, and stored in 70% alcohol before rehydrating and staining DNA by the Feulgen technique. Intestinal crypts in controls and test samples were separated by microdissection in 45% acetic acid, squashed under a coverslip and examined by light microscopy. The numbers of metaphase arrests accumulating over 3 h in 15 crypts were counted in control and test specimens, and means and SEs determined. Statistical analysis was done by paired t-test. The mean

SIR-Mepham

concern

815

numbers of metaphase arrests/crypt in controls was 5’8 (SE ’0-18) and 12-9 in tests (0-71) (p<0001), showing that 400 ng/mL of rhIGF-1 had a mitogenic effect on the adult human duodenal mucosa cultured in vitro. Cows’ milk contains 4 ng/mL IGF-1 (range 1-8 ng/mL) and BST-milk up to 50 ng/mL IGF-1. The concentrations of rhIGF-1 (400 ng/mL) we used were higher than these values, but lower concentrations (100 and 200 ng/mL) also increased crypt epithelial cell proliferation in preliminary dose-response studies. The combination of IGF-1 in BSTmilk and IGF-1 normally secreted into the human gastrointestinal lumen2 would augment intraluminal

concentrations of this hormone, increasing the local mitogenic effects on gut tissues. DN

possibility of

Challacombe, E E Wheeler

Somerset Children’s Research Unit, Taunton and Somerset Hospital, Musgrove Park Branch, Taunton TA1 5DA, UK

1

2

Challacombe DN, Wheeler EE. Trophic action of epidermal growth factor on human duodenal mucosa cultured in vitro. Gut 1991; 32: 991-93. Chaurasia OP, Marcuard SP, Seidel ER. Insulin-like growth factor I in human gastrointestinal exocrine secretions. Regul Pept 1994; 50: 113-19.

report contains several inaccuracies and inconsistencies that need to be addressed. They state "This ... suggests a possibility of more than tenfold increase in milk IGF1concentration". In fact, the joint WHO/FAO expert committee on food additives concluded after reviewing the IGF-1 data sets of all four companies developing a somatotrophin product for use in dairy cattle "Some studies suggest that rbST treatment may produce a slight increase in the average IGF-1 concentration; however, the most definitive and comprehensive studies show that IGF-1 concentrations are not altered after rbST treatment".1 They also say that FDA data showed some statistically significant systemic effects of orally-administered IGF-1 in rats which, remarkably, Juskevich and Guyer discounted as sporadic results. However, they go on to indicate "at concentrations of IGF1 in milk it is unlikely that systemic effects would be induced". This statement is in agreement with their reference to Olanrewaju,2 who conclude that they could not induce a systemic effect with local intraileal infusion of IGF-1 and who cites work showing that 180 µg of met-IGF-1 per day given as continuous subcutaneous infusion was needed to produce a significant effect on body weight in hypophysectomised rats: at a milk concentration of 4 ng/mL this equates to a daily milk intake per rat of 45 L, and all the IGF-1 in that milk must escape proteolysis

SIR-Mepham and colleagues’

during digestion. Mepham and co-workers cite as a concern "epidermal growth factor, which like IGF1 has biological effects on human gut". Epidermal growth factor is undetectable in bovine milk4 and there is no evidence that it is increased with BST treatment of lactating cows. Also, although systemic effects of IGF-1 on gut tissues in rats have been recorded we are not aware that oral IGF-1 has been shown to have effects on human gut. They then cite Olanrewaju et al2 as saying that "intraluminal infusion of IGF1 in rats, at concentrations equivalent to those in bovine milk, increase cellularity of the intestinal mucosa". In this model, rats were fitted with intraperitoneal osmotic minipumps which infused 10 nmol/L per hour IGF-1 or saline into the lumen of the ileum for 66 h. During that period animals were not fed and lost 27-34% of body weight. Food consumption stimulates both the volume and IGF-1 concentrations of exocrine secretions

816

of the digestive tract’ resulting in IGF-1 exposure at the small intestine well in excess of the doses used in this study. Since there was no sham-operated fed control it is not possible to determine the potential negative effects that feed withdrawal had on the indices measured in the controls. Additionally, we cannot establish if lower IGF-1 concentrations in the gut of the treated group sensitise the tissue to respond to IGF-1 infusion. Therefore, any effects of infused IGF-1 may well be undetectable in fed treated and control animals. Furthermore, since IGF-1 concentration in milk of rBSTtreated cows is unchanged the issue is moot. The proliferative response in tissue culture of human intestinal and rat muscle cell lines and bovine mammary epithelial primary cells to milk from BST-treated cows has been studied with no detectable change in mitogenesis.’5 These cell lines collectively respond to IGF-1, IGF-2, insulin, fibroblast growth factor, epidermal growth factor, and transforming growth factor alpha. Thus, there is no evidence that hormonal content of milk from BST-treated cows is in any way different from cows not so treated. Robert J Collier, David R Clemmons, Sharon M Donovan Monsanto Company, St Louis, MO 63198, USA; Department of Medicine, University of North Carolina, Capel Hill, NC; and Division of Nutrition, College of Agriculture, University of Illinois, St Louis, MO

1

2

3 4

5

Fortieth report of the Joint FAO/WHO Expert Committee on Food Additives. Evaluation of certain veterinary drug residues in food. WHO Tech Rep Ser 1993; 832: 41. Olanrewaju H, Patel L, Seidel ER. Trophic actin of local intraileal infusion of insulin-like growth factor I: polyamine dependence. Am J Physiol 1992; 263: E282-86. Donovan SM, Odle J. Growth factors in milk as mediators of infant development. Ann Rev Nutr 1994; 14: 147-67. Chaurasia OP, Marcuard SP, Seidel ER. Insulin-like growth factor I in human gastrointestinal exocrine secretions. Reg Peptides 1994; 50: 113-19. McGrath MF, Eppard PJ, Sweeny CA, Keller PG, Collier RJ. Proliferative activity of bovine milk following Sometribove (methionylbovine somatotropin, rbST) administration. J Dairy Sci 1993; 76

(suppl):

1-170.

SIR-Mepham and colleagues cite Lilly Industries as stating that milk from cows receiving BST is "unlikely to contain more than 50 ng/mL IGF1", which they compared with a bulk tank average of 4 ng/mL on the basis of Juskevich and Guyers report’ to support speculation that the use of BST in dairy cows might raise the concentration of IGF-1 in milk tenfold. Firstly, the reference they give for the statement attributed to Lilly is incorrect; Lilly did not make an application to the CEC Committee for Veterinary Medicinal Products during 1993. Lilly Industries did use an illustration in documents presented to the UK Veterinary Products Committee and Medicines Commission, in support of food safety, which referred to a hypothetical level of 50 ng/mL IGF-1 in milk. The context at the time was a discussion of a study in which hypophysectomised rats (which are very sensitive to IGF-1 given parenterally) were gavaged with IGF-1 at doses up to 1000 µg/kg body weight daily. Oral IGF-1 had no effect on growth, Rats

organ

weights, haematology,

killed

or serum

chemistry.

the end of the study and histopathological testing was done on all major organs and on the various regions of the intestinal tract. The gut sections were reviewed by two independent experts, in addition to the study pathologists. Oral IGF-1had no effect on the histology of any region of the gut. It is customary to calculate safety margins for human use on the basis of sensitive animal models. To be conservative in calculating a safety margin for IGF-1, 50 ng/mL was selected as the maximum reported for individual cows, whether receiving bST or not. On this basis a 20 kg child were

at

drinking 1 L per day would take in 50 ng, or 2-5 yg IGF1/kg body weight. 400 times this amount had no observable effect on the gut or any other organ in hypophysectomised rats.

The concentration of 4 ng/mL for untreated cows which Mepham and colleagues selected’ was the average result for samples of 100 bulk tanks. Each tank would have contained the mixed milk from many cows. Juskevich and Guyer’ cite a study in which the maximum concentration in individual cows was 30-5 ng/mL and another in which the average for a group of four cows was 28-4 ng/mL; none of these cows had

received bST. As these workers state, the concentration of IGF-1 in milk varies widely between animals and it is determined by stage of lactation and parity. Increases in IGF-1 in the milk of cows receiving BST are small in the context of this natural variation. Consumers generally receive milk pooled from a large number of cows, so the concentration of IGF-1 will be much less than 50 ng/mL and the safety margin will be correspondingly wider. Lilly has no data that could be construed to support anything approaching the tenfold increase suggested in the letter of Mepham and colleagues. Mepham and colleagues imply that milk proteins might protect IGF-1 from digestion by proteolytic enzymes. Lilly’s safety evaluation included a test in which the milk of cows receiving more than seven times the recommended dose of BST was given to hypophysectomised rats for 10 days. There were no effects on growth or epiphyseal cartilage development. No regulatory authority has ever concluded that there is a risk to human health from milk from cows receiving bST. They include the UK Veterinary Products Committee and Medicines Commission, the EC Committee for Veterinary Medicinal Products, the US Food and Drug Administration and Joint WHO/FAO Committee on Food Additives.2

26 cases of salivary gland tumours, focal staining for both PSAP and PSA was seen. These findings show that the combined PSAP and PSA staining of adenocarcinomas is not specific for prostate carcinoma. Because of the remarkable regression of tumour after treatment with goserelin (which decreases testicular testosterone), we postulated that this effect is mediated through androgen receptors. Preliminary data on expression of androgen receptor’ in salivary gland tumours show the presence of androgen receptors in adenomas and carcinomas. Further data are required to establish whether androgen receptor staining in parotid tumours may be clinically useful as an indicator of susceptibility to hormonal treatment.

R W M van der Hulst, J H J M van Krieken, Th H J J Gerritsen, R J Baatenburg de Jong, A A B Lycklama à Nijeholt, A E Meinders

van

der Kwast,

Department of General Internal Medicine, University Hospital Leiden, p2300 RC Leiden, Netherlands, Laboratory of Pathology, University Hospital Leiden; Erasmus University Rotterdam, Department of Otolaryngology and Head and Neck Surgery, and Department of Urology, University Hospital, Leiden

1

Brawer MK. Prostate specific 161-68.

2

Allhoff EP, Proppe KH, Chapman CM,

3

4

5

antigen:

a

review. Acta Oncol et

1991; 30:

al. Evaluation of prostate

specific acid phosphatase and prostate specific antigen in identification of prostatic cancer. J Urol 1983; 129: 315-18. Yam LT, Winkler CF, Janckila AJ, et al. Prostatic cancer presenting as metastatic adenocarcmoma of undetermined origin. Cancer 1983; 51: 283-87. Krieken

JHJM. Prostate marker immunoreactivity in salivary gland neoplasms. A rare pitfall in immunohistochemistry. Am J Surg Pathol 1993; 17: 410-14. Ruizeveld de Winter JA, Trapman J, Vermey M, et al. Androgen receptor expression in human tissues: an immunohistochemical study. J Histochem Cytochem 1991; 39: 927-36.

J I D Wilkinson Lilly Industries, Chapel Hill, Basingstoke,

Hants RG21 2SY, UK

1 Juskevich JC, Guyer CG. Bovine growth hormone: human food safety evaluation. Science 1990; 249: 875. 2 Joint FAO/WHO Expert Committee on Food Additives. 40th meeting, Geneva, June 9-18, 1992.

Partial remission of parotid gland carcinoma after goserelin SIR-We describe partial remission of a parotid gland carcinoma treated with goserelin, a luteinising hormonereleasing hormone analogue commonly used for metastatic prostate carcinoma. The patient presented with a locally invasive, moderately differentiated adenocarcinoma of the parotid gland. Biopsy samples from the tumour showed positive staining for prostatic serum acid phosphatase (PSAP) and prostatespecific antigen (PSA) markers. This staining is regarded as specific for prostate carcinoma.1-3 In our patient no primary prostate carcinoma was detected despite extensive radiological examination and histological examination of biopsy samples from the prostate. Nonetheless, we treated him with goserelin 3-7 mg per month subcutaneously for 3 months, which resulted in partial remission of the parotid tumour mass. Because of the discrepancy between clinical and immunohistochemical findings, we assessed the specificity of PSAP and PSA antibody staining in salivary gland tumours and in normal salivary glands.4 In normal salivary gland tissue, staining for PSA alone was positive in 17 out of 18 cases, whereas staining for both PSAP and PSA was negative in all 18 biopsy samples. By contrast, in 10 of

L-tryptophan and eosinophilia-myalgia syndrome SIR-In their reply accompanying my letter (April 23, p 1035-36) Eidson et al deny that there are only two published case-control studies-theirs’1 and another appearing as a single paragraph2-that show a purported causal link between L-tryptophan and the eosinophiliamyalgia syndrome (EMS). In the additional studies they cite/3 that relation was not examined. Instead, it was claimed that causation was now established, and the further question was whether a contaminant accounted for it. The additional studies could not determine whether there was an overall association since they were confined to cases and controls who used L-tryptophan. Eidson et al do not respond to my point that "a test for an association suspected because of a cluster must be independent" of that cluster. Not only were 2 members of a previously reported cluster’ improperly included in their study but also 6 additional cases were reported to the New Mexico Department of Health from Nov 7 to Nov 9, 1989, before the study began. Their inclusion again violates the principle that a study mounted to confirm a suspicion must be independent of the data that gave rise to that suspicion. Eidson et al stated’ that they excluded cases with any mention "at any period during the patient’s lifetime" of allergic rhinitis or drug hypersensitivity in the "medical record or subsequent interviews" (including questionnaires, my inference). They now state that they relied on contemporaneous "hospital or clinic charts at the facility that had requested the blood counts". After completion of the study they obtained additional records in which they "do find mention of allergies for some of the ... cases". 817