Safety evaluation of a hexose oxidase expressed in Hansenula polymorpha.

A programme of studies was conducted to establish the safety of hexose oxidase (HOX) from Chondrus crispus expressed in the yeast Hansenula polymorpha to be used as a processing aid in the food industry. Rat feeding studies were conducted to assess acute and subchronic oral toxicity. In addition, the potential of the enzyme to cause mutagenicity and chromosomal aberrations was assessed in microbial and tissue culture in vitro studies. Acute and subchronic oral toxicity was not detected at the highest dosage recommended by OECD guidelines. There was no evidence of mutagenic potential or chromosomal aberrations. The no-observed-adverse-effect level (NOAEL) derived from the 13-week study was 5000 units/kg body weight/day. In conclusion it can be considered a safe processing aid for use in the food industry.

A pneumatic device for rapid loading of DNA sequencing gels.

This work describes the design and construction of a device that facilitates the loading of DNA samples onto polyacrylamide gels for detection in the Perkin Elmer/Applied Biosystems (PE/ABI) 373 and 377 DNA sequencing instruments. The device is mounted onto the existing gel cassettes and makes the process of loading high-density gels less cumbersome while the associated time and errors are reduced. The principle of operation includes the simultaneous transfer of the entire batch of samples, in which a spring-loaded air cylinder generates positive pressure and flexible silica capillaries transfer the samples. A retractable capillary array carrier allows the delivery ends of the capillaries to be held up clear of the gel during loader attachment on the gel plates, while enabling their insertion in the gel wells once the device is securely mounted. Gel-loading devices capable of simultaneously transferring 72 samples onto the PE/ABI 373 and 377 are currently being used in our production sequencing groups while a 96-sample transfer prototype undergoes testing.

Traumatic optic neuropathy. A meta-analysis.

BACKGROUND: The management of traumatic optic neuropathy remains controversial. Reports of improvement have been published after observation alone, treatment with corticosteroids and surgical decompressions. OBJECTIVE: To systematically review the published literature about traumatic optic neuropathy using a meta-analysis. METHODS: We performed a retrospective literature review of case series and case reports of traumatic optic neuropathy. They include all English language cases and selected non-English language cases for which patient data were available. The cases were organized into four grades based on visual acuity and the locations and type of fracture. Grade 1 included patients with visual acuity greater than 20/200 in the affected eye and without a posterior orbit fracture; grade 2, patients with visual acuity between 20/200 and light perception; grade 3, patients without light perception or with a nondisplaced posterior orbital fracture and remaining vision; and grade 4, patients with no light perception and a displaced posterior orbital fracture. A meta-analysis was performed, analyzing for each case the recovery of visual acuity for treatment, fracture pattern, and grade. RESULTS: The recovery of vision in treated patients was significantly better than the recovery in patients receiving no treatment. No significant difference in improvement was found among patients treated with corticosteroids alone, with surgical decompression alone, or with corticosteroids and surgical decompression. Recovery was related to the severity of initial injury, as reflected in the grading system. A trend was noted for better improvement of visual acuity in patients without orbital fractures than those with orbital fractures, and also in patients with anterior orbital fractures than in patients with posterior fractures. CONCLUSIONS: Treatment with corticosteroids, extracranial decompression, or both, is better than no treatment of traumatic optic neuropathy. Because the data are insufficient to determine whether corticosteroids, surgery, or the use of both treatments is most effective, the findings of the ongoing International Optic Nerve Trauma Study should prove valuable. The standardized grading system we developed is a useful tool for comparing studies and treatment protocols.

The effect of mono-(2-ethylhexyl) phthalate and other phthalate esters on lactate production by Sertoli cells in vitro.

Sertoli cells produce lactate and pyruvate as energy substrates for the developing germ cells in the testis. Since the Sertoli cells are thought to be the initial target for phthalate esters causing testicular atrophy, the effect of some phthalates on lactate and pyruvate production by primary Sertoli cell-enriched cultures was studied. Mono-(2-ethylhexyl) phthalate (0.1-200 microM) produced a concentration-dependent stimulation of lactate, but not pyruvate production over a 24 h treatment period and an increase in the ratio of lactate/pyruvate concentration in the culture medium. Di-(2-ethylhexyl) phthalate and 2-ethylhexanol (200 microM) had no such effects. Other phthalate monoesters known to cause testicular atrophy also increased Sertoli cell lactate production and the lactate/pyruvate ratio, whereas monoesters not associated with testicular damage in vivo had no such effects. The results suggest that loss of germ cells in phthalate-induced testicular atrophy is not due to inhibition of energy substrate production by the Sertoli cells and that stimulation of lactate production may be a useful in vitro marker for phthalate esters and related compounds that cause testicular injury.

Studies on the metabolism of deoxynivalenol in the rat.

The metabolism and tissue distribution of [14C]deoxynivalenol have been studied in male PVG rats. Following administration of a single oral 10-mg/kg dose, radioactivity excreted in the urine and faeces accounted, respectively, for 25 and 64% of the administered dose within 96 hr. Less than 0.15% of the dose was detected in the respired air. Very little radioactivity appeared to be retained in any of the tissues examined after 96 hr. HPLC separation of several urinary and faecal metabolites was achieved on a reversed-phase column, using two different elution systems, one at neutral pH and one acidified. Two of the major non-polar HPLC peaks were identified by gas chromatography-mass spectrometry as unchanged deoxynivalenol and 3 alpha,7 alpha,15-trihydroxytrichothec-9,12-dien-8-one.

The role of metabolism in 2-methoxyethanol-induced testicular toxicity.

The role of metabolism in 2-methoxyethanol (ME)-induced testicular toxicity has been investigated with Sprague-Dawley rats. Following administration of [14C]ME (250 mg/kg, ip) to a group of animals, there was evidence of testicular damage, identified as depletion of the spermatocyte population. Radioactivity detected in urine over 48 hr after treatment accounted for 55% of the dose. The major urinary metabolites were identified by HPLC and isotope dilution analysis, as methoxyacetic acid (MAA) and methoxyacetylglycine (accounting for 50 to 60% and 18 to 25%, respectively, of urinary radioactivity). Analysis of plasma revealed a rapid conversion of ME to MAA (t1/2 for disappearance of ME = 0.6 +/- 0.03 hr) and gradual clearance of radioactivity (t1/2 = 19.7 +/- 2.3 hr). Pretreatment of animals with pyrazole (400 mg/kg, ip) 1 hr prior to [14C]ME dosing gave complete protection against the testicular toxicity of ME. Radioactivity detected in the urine from the pyrazole-pretreated groups over 48 hr (18%) was significantly lower than in the ME-only group. The major radioactive peak co-chromatographed with ME (30 to 36% of the total urinary radioactivity). MAA and methoxyacetylglycine were not major metabolites. Analysis of plasma revealed almost complete inhibition of the conversion of ME to MAA (t1/2 for disappearance of ME = 42.6 +/- 5.6 hr, clearance of radioactivity t1/2 = 51.0 +/- 7.8 hr). The results demonstrate that metabolic activation is required for 2-methoxyethanol to exert toxicity to the male reproductive system.

Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat.

Ethylene glycol monomethyl (EGM) and monoethyl (EGE) ethers were administered po to rats at dosages varying from 50 to 500 mg/kg body weight/day for EGM and 250 to 1000 mg/kg body weight/day EGE for 11 days. First evidence of testicular damage following EGM treatment was observed 24 hr after a single dose of 100 mg/kg body weight when the lesion appeared localized in the primary spermatocyte. At 16 hr after a single dose of 500 mg/kg, mitochondrial damage was one of the first subcellular changes to be demonstrated. Treatment of animals with EGE resulted in a similar lesion; however, to obtain damage of equivalent severity, a larger dosage for a longer period was required. In limited studies with 2-methoxy- and 2-ethoxyacetic acids (putative metabolites of EGM and EGE, respectively), using equimolar doses to their parent compounds (500 mg EGM or EGE/kg for 4 or 11 days, respectively) gave damage of equivalent severity to the corresponding glycol ether. After dosing animals with 500 mg EGM/kg body weight for 4 days, the testes recovered weight, and the majority of tubules recovered their spermatogenic potential within one full maturation cycle. The recovery study also indicated a possible effect on the spermatogonia in a small number of tubules although no morphological abnormalities to this cell type could be observed. No effect levels over the 11-day treatment period were 50 and 250 mg/kg body weight/day for EGM and EGE, respectively.

Effect of DI-n-pentyl phthalate treatment on testicular steroidogenic enzymes and cytochrome P-450 in the rat.

Treatment of young male rats with dipentyl phthalate (DPP) produced significant decreases in testicular cytochrome P-450, cytochrome P-450 dependent microsomal steroidogenic enzymes (17 alpha-hydroxylase, 17-20 lyase) and in the maximal binding of a natural substrate (progesterone) to testis microsomes. No effect was demonstrated by this compound on hepatic cytochrome P-450 content. Treatment of animals with a phthalate ester not causing testicular atrophy (diethyl phthalate; DEP) produced no significant changes in any of the parameters measured. This effect on the enzymes responsible for androgen production may be important as a mechanism of action involved in the development of phthalate-induced testicular damage.

Differences in urinary metabolic profile from di-n-butyl phthalate-treated rats and hamsters. A possible explanation for species differences in susceptibility to testicular atrophy.

A high-performance liquid chromatographic method for the direct analysis of the urinary metabolites of di-n-butyl phthalate (DBP) is described. In both rats and hamsters the major urinary metabolite found after treatment with either DBP or mono-n-butyl phthalate (MBP) was MBP glucuronide and not MBP as previously reported. The levels of unconjugated MBP in the urine of animals treated with DBP or MBP were three- to fourfold higher in the rat than in the hamster. However, intestinal esterase activities were comparable in the two species, whereas the activities of testicular beta-glucuronidase were significantly higher in rats compared to hamsters. It is possible that the differences in the concentration of free MBP, a substance known to produce testicular damage directly in the rat in vitro, may account for the lack of injury seen in hamsters after oral treatment with either DBP or MBP.

Studies on the testicular effects and zinc excretion produced by various isomers of monobutyl-o-phthalate in the rat.

Mono-n-butyl, -iso-butyl, -sec-butyl or -tert-butyl esters of o-phthalic acid were orally administered at 800 mg/kg body wt./day for 6 days, to young male rats. All of the animals except those treated with the mono-tert-ester developed marked testicular atrophy. Additionally, only those isomers producing testicular damage were found to alter zinc metabolism by increasing the urinary excretion of zinc and by depleting the concentration of this element in testicular tissues.