Toxicity, occurrence and negative effects of PR toxin – the hidden enemy by Katia Pedrosa and Karin Griessler, O mous epithelioma and a uterine sarcoma, Biomin Holding, Industriestrasse 21, O which were shown histologically. Tests with 3130 Herzogenburg, Austria. Salmonella typhimurium, Saccharomyces cerevisiae and Neurospra crassa also he PR toxin is a secondary metabolite O O showed mutagenicity from PR toxin expo - of roqueforti. It has long O sure. Tbeen reported that poorly ensiled Toxic effects in mice and rats have crops, which promote the growth of included abdominal writhing, decreased Penicillium moulds, pose a serious threat to O H motor activity and respiratory rate, weak - the health and productivity of animals and ened hind legs and ataxia. In mice, rats and result in significant economic losses. Fig. 1. Chemical structure of PR toxin. cats PR toxin given ip caused ascites fluid This article reviews the present knowledge and oedema in the scrotum and lungs. on the toxicity, occurrence and negative important role in biological activity. The Intravenous (iv) injection caused oedema effects of PR toxin in animals. biosynthesis of PR toxin has been studied in the lungs, giving rise to a large volume of Ensiling or fermenting forages with lactic with both 14C- and 13C-labelled precursors pleural and pericardial fluid. Injecting rats acid producing bacteria is a relatively simple and indicates that PR toxin is formed via an with LD50 doses (11.6mg/kg, ip and yet effective way of preserving forage for isoprene biosynthetic pathway. 8.2mg/kg, iv), resulted in an increase in the future use by livestock. Eremofortin C (EC) (another secondary water content of the lungs but a decrease in One of the spoilage fungi most commonly metabolite) and PR toxin are closely related the skin. found in silages as well as other habitats with to each other and differ only by a hydroxyl Still et al. (1972) and Chen et al. (1982) limited oxygen is , and functional group in EC and an aldehyde func - conclude that PR toxin produced acute under such improper ensiling conditions, tional group in PR toxin at the C-12 position toxic effects in animals via an increase of spoilage by Penicillium as well as Aspergillus responsible for the conversion of EC into capillary permeability and a direct damage moulds that were present in the feedstuff at PR toxin. In contrast to PR toxin, EC does to the lungs, heart, liver and kidneys. harvest is likely to occur accompanied with not show significant toxicity in animals. These researchers also suspected PR toxin some degree of contamination. was a vector in a case study where abortion One of the of concern that is and retained placenta occurred. produced by Penicillium roqueforti is PR Toxicity Little information, some of which is con - toxin. The effects of long term PR toxin troversial, has been reported about PR toxi - exposure are a subject of interest since PR toxin has been shown to be lethal to ani - city in dairy cows. ruminant diets frequently contain a high pro - mals and exhibits a broad spectrum of bio - Vesely et al. (1981) fed dairy cows maize portion of forages (dairy cows eating chemical activities, which cause toxicoses in silages infected with Penicillium roqueforti 15±kg/dry matter/day) such as grass or animals. and reported the animals had loss of maize silage, hay and straw. High producing Up to now, studies with laboratory ani - appetite, cessation of rumen activity, and livestock, especially, can be subject to sub- mals report that PR toxin is considered to gastroenteritis. acute symptoms of PR toxin mycotoxicosis. be the most toxic of the P. roqueforti myco - Scudamore and Livesey (1998) reported Besides PR toxin, other toxins such as toxins. This toxin is lethal to rats and mice that cows aborted their first calves in the roquefortine C, and mycophenolic by either oral or intraperitoneal (ip) admin - seventh and eighth month of pregnancy, but acid can be produced by this . istration. LD50 values in mice ranged from they cautioned about solely attributing these Auerbach et al. (1998) found P. roqueforti 1.0-5.8 mg/kg (ip) and 58-100mg/kg/BW symptoms to PR toxin since Penicillium in 89% of visibly mouldy samples and in 85% orally. roqueforti is known to be capable of form - of visibly unmouldy samples rendering visible PR toxin causes degenerative changes in ing a number of other toxic metabolites. inspection essentially useless in identifying livers and kidneys of rats. Wei and Liu More recently, Nielsen et al. (2006) Penicillium mould contamination of forages. (1978) verified inhibition of in vivo protein observed that cattle consuming Penicillium synthesis in rat liver, probably because the roqueforti contaminated feed had variable toxin prevents the initiation and elongation toxic outcomes with some animals exhibit - Chemical structure steps of transcription. In addition to the ing severe symptoms including haemorrhage inhibitory effects on proteins, this mycotoxin and death. The PR toxin molecule contains several has also revealed strong inhibitory effects on Current data suggest that roquefortine C functional groups including an aldehyde, an ribonucleic acid and deoxyribonucleic acid is not a very toxic mycotoxin in animals. This and also the fact that roquefortine C is acetoxy, and an α, β-unsaturated ketone biosynthesis of Ehrlich ascites tumour cells. group, in addition to two stable epoxide Carcinogenic activity has also been shown produced over a broad range of environ - rings (Fig. 1). to result from PR toxin exposure. Rats fed mental conditions lead Nielsen et al. (2006) The aldehyde group appears to play an PR toxin developed adenocarcinoma, squa - Continued on page 9

International Dairy Topics — Volume 9 Number 2 7 Continued from page 7 P. roqueforti P. paneum P. cameum to hypothesise that the seemingly well known acute toxicities associated with P. Roquefortine C Roquefortine C Roquefortine C roqueforti contamination were due to PR Roquefortine D Marcofortines A-C Festuclavine as P. crustosum toxin or compounds other than roquefor - as P. roqueforti tine C. Despite the symptoms reported by Roquefortines A and B Patulin Roquefortines A and B as these authors, Sumarah et al. (2005) stated P. crustosum that PR toxin does not seem sufficiently toxic on its own to explain symptoms such PR-toxin Orsellinic acid as P. roqueforti Mycophenolic acid as general ill-thrift, abortions and more Eremofortins A-E Botryodiplodin as P. roqueforti Patulin as P. roqueforti and severe toxic signs. as P. roqueforti var. Cameum Indeed, there is little information on the toxic effects of PR toxin in dairy cows and PR-imine Penicillic acid as P. suavolens therefore more research is needed regard - and as P. roqueforti ing whether PR toxin does in fact cause PR-amide Cycoplastic acid problems in dairy cows and at which conta - Mycophenolic acid mination levels. This need for more research is highlighted Table 1. Mycotoxins and other secondary metabolites from Penicilliums (Nielsen et in a recently published review about myco - al., 2006). toxins in silages in which Storm et al. (2008) state that the toxic effects of PR toxin in detected although lumps of infected silage were capable of forming the mycotoxins ruminants are still unknown. contained several unidentified fungal metab- mycophenolic acid (MPA), patulin (PAT), olites. penicillic acid (PA) or PR toxin (PRT). In contrast, Yu et al. (1999) analysed 63 MPA, PAT, PA and PRT levels above the Occurrence of PR toxin samples (25 hay and 38 other feedstuffs, detection limit were measured for the first including corn silage and mixed feed) and time at 36, 22-27, 13, and 49 days of incuba - O’Brien et al. (2006) analysed secondary showed that the frequency of PR toxin in tion, respectively.As storage times increased metabolites produced by Penicillium these samples was 76% with an average toxin concentrations decreased to a low or paneum and Penicillium roqueforti from contamination of 130ppb. non-detectable level. baled grass silage. Therefore, in light of results such as these Therefore, the researchers, Müller and A total of 157 isolates were investigated, PR toxin has been suggested as the respon - Amend (1997), concluded that the probabil - comprising 78 P. paneum and 79 P. roque - sible agent for problems resulting from feed - ity of detecting these mycotoxins in P. forti isolates randomly selected from more ing mouldy corn silage. Surveys of grass and roqueforti moulded maize silage is low than 900 colonies cultured from the silage corn silage in Europe reported an occur - under practical conditions of agriculture dur - bales. Roquefortine C, marcfortine A, and rence of P. roqueforti in up to 40% of sam - ing the growth phase of this fungus and again andrastin A were consistently produced, ples, which was associated with cattle after prolonged storage. whereas PR toxin and patulin were not. disorders, but the occurrence or levels of Although the knowledge about PR toxin is These findings mostly agreed with the liter - PR toxin was not reported. still low, it is hypothesised that the reaction ature as noted in Table 1. of this toxin with silage contents (for exam - Roquefortine C and mycophenolic acid are ple NH3) may explain the low toxicity. This the two most frequently detected mycotox - Stability of PR toxin could also account for these compounds ins produced by the P. roqueforti group in quickly becoming undetectable in the occur - silages (see photograph below), whereas PR Penicillium roqueforti requires amino nitro - ring substrate. toxin and patulin have been detected only gen for metabolite formation, and the toxins occasionally owing to their unstable nature it produces can be formed under conditions in this matrix. of low oxygen (20-30% saturation). Conclusions Penicillium roqueforti was the predomi - Even though PR toxin is of greater toxico - nant fungus found during a study of spoiled logical concern based on studies with labo - Investigations with laboratory animals maize silage carried out in the Netherlands ratory animals, the toxin is known to be regarding the toxicity of PR toxin indicated between 1986-1990. No PR toxin was unstable and to occur bound to amino acids, that this mycotoxin is considered the most amines and NH3. In toxic amongst P. roqueforti toxins. How- another study, Chang ever, the effects of PR toxins on dairy cows Penicillium roqueforti in maize silage. et al. (1993), reported are still largely unknown as very few studies that PR toxin was trans - with PR toxin and dairy cows are available. formed into PR-imine Additionally, due to fact that PR toxin is when PR toxin was not stable during storage and may react with incubated with the cul - silage constituents, the toxicity may be very ture medium during 37 low except when recently formed concen - days, and PR toxin can trations are high. also be degraded into The detection of these toxins in silages PR acid. Thus, these under practical conditions is also difficult, researchers proposed again possibly due to their short life and the that PR toxin is apparent instability. On a practical point of degraded into PR-imine view, the usual presence of more than one and PR-amide in the mycotoxin in silages may be a more accu - culture medium of P. rate explanation for the problems often roqueforti. found in dairy cattle rather than the pres - Müller and Amend ence of PR toxin alone. I (1997) inoculated maize silage with Penicillium References are available roqueforti strains which from the authors on request

International Dairy Topics — Volume 9 Number 2 9